Keratinocyte growth factor-2 formulations

ABSTRACT

The invention is directed to liquid and lyophilized forms of Keratinocyte Growth Factor-2 (KGF-2) and derivatives thereof. This invention further relates to the formulation of KGF-2 for therapeutic use, for example, to promote or accelerate wound healing.

[0001] This application claims the benefit of priority of the filingdate of Provisional Application No. 60/068,493 filed on Dec. 22, 1997,which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention is directed to liquid and lyophilizedformulations of Keratinocyte Growth Factor-2 (KGF-2) and derivativesthereof. This invention further relates to formulations of KGF-2,especially topical and injectable formulations, that can be employed fortherapeutic use in indications requiring soft-tissue growth andregeneration.

[0004] 2. Related Art

[0005] The fibroblast growth factor family has emerged as a large familyof growth factors involved in soft-tissue growth and regeneration. Itpresently includes several members that share a varying degree ofhomology at the protein level, and that, with one exception, appear tohave a similar broad mitogenic spectrum, i.e., they promote theproliferation of a variety of cells of mesodermal and neuroectodermalorigin and/or promote angiogenesis.

[0006] KGF was originally identified as a member of the FGF family bysequence homology or factor purification and cloning. Keratinocytegrowth factor (KGF) was isolated as a mitogen from a cultured murinekeratinocyte line (Rubin, J. S. et al., Proc. Natl. Acad. Sci. USA86:802-806 (1989)). Unlike the other members of the FGF family, it haslittle activity on mesenchyme-derived cells but stimulates the growth ofepithelial cells. Keratinocyte growth factor is produced by fibroblastsderived from skin and fetal lung (Rubin et al. (1989)). The Keratinocytegrowth factor mRNA was found to be expressed in adult kidney, colon andilium, but not in brain or lung (Finch, P. W. et al. Science 245:752-755(1989)). KGF displays the conserved regions within the FGF proteinfamily. KGF binds to the FGF-2 receptor with high affinity.

[0007] Impaired wound healing is a significant source of morbidity andmay result in such complications as dehiscence, anastomotic breakdownand, non-healing wounds. In the normal individual, wound healing isachieved uncomplicated. In contrast, impaired healing is associated withseveral conditions such as diabetes, infection, immunosuppression,obesity and malnutrition (Cruse, P. J. and Foord, R., Arch. Surg.107:206 (1973); Schrock, T. R. et al., Ann. Surg. 177:513 (1973); Poole,G. U., Jr., Surgery 97:631 (1985); Irvin, G. L. et al., Am. Surg. 51:418(1985)).

[0008] Wound repair is the result of complex interactions and biologicprocesses. Three phases have been described in normal wound healing:acute inflammatory phase, extracellular matrix and collagen synthesis,and remodeling (Peacock, E. E., Jr., Wound Repair, 2nd edition, W BSaunders, Philadelphia (1984)). The process involves the interaction ofkeratinocytes, fibroblasts and inflammatory cells at the wound site.

[0009] It is desirable to formulate polypeptides that are capable ofpromoting and enhancing soft-tissue growth and regeneration inpharmaceutical compositions that (1) are stable over prolonged periodsof storage, (2) increase the pharmacological activity or effectivenessof the the polypeptide and/or (3) allow facile application oradministration of the polypeptide in therapeutic regimens.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to liquid and lyophilizedformulations of KGF-2 and deletion or point or substitution mutantsthereof (referred to herein as KGF-2 polypeptides). This inventionfurther relates to the use of such formulations of KGF-2 polypeptides topromote or accelerate soft tissue growth or regeneration, for example inwound healing, or in treating mucocytis or inflammatory bowel disease.Preferred formulations of the present invention employ novel mutantforms of KGF-2, and in one embodiment employ a deletion mutant referredto herein as KGF2-Δ33. The co-ingredients employed in the formulations(1) provide storage stability to the KGF-2 polypeptide, (2) furtherenhance soft-tissue healing activity of the therapeutic composition,and/or (3) provide the KGF-2 polypeptide in an active form whileallowing facile application and administration for particulartherapeutic purposes.

[0011] A first aspect of the present invention relates to a formulationcomprising a KGF-2 polypeptide and a buffering agent having a bufferingcapacity of between about pH 5.0 and about pH 8.0. Useful buffersinclude phosphate, acetate, aconitate, succinate, malate, carbonate andcitrate buffers, citrate being preferred.

[0012] A second aspect of the invention relates to a formulationcomprising a KGF-2 polypeptide, a lyophilization bulking agent and abuffering agent having a buffering capacity of between about pH 5.0 andabout pH 8.0. Useful buffers include phosphate, aconitate, succinate,malate, carbonate and citrate buffers, citrate being preferred.

[0013] A third aspect of the invention relates to a formulationcomprising a KGF-2 polypeptide and a thiol-containing compound,preferably monothioglycerol, capable of stabilizing the KGF-2polypeptide. This formulation preferably includes a buffering agenthaving a buffering capacity of between about pH 5.0 and about pH 8.0.This formulation may also include one or more antioxidants and or one ormore metal chelating agents.

[0014] A fourth aspect of the present invention relates to a formulationcomprising a KGF-2 polypeptide, a buffer, and a high molecular weightcompound that causes the formulation to gel at a certain predefinedtemperature. A preferred high molecular weight compound is a Pluronic orPoloxamer polyoxyethylene-polyoxypropylene block copolymer. Athiol-containing compound, such as monothioglycerol, can be included inthe formulation to provide added stability to the polypeptide.

[0015] A fifth aspect of the present invention relates to a formulationcomprising a KGF-2 polypeptide, a buffering agent and a thickeningagent. Thickening agents are used to increase the viscosity of theformulation. Preferred thickening agents are carboxymethyl cellulose(CMC), hydroxyethyl cellulose (HEC), hydroxypropylmethyl cellulose(HPMC), Natrosol, and Carbomers.

[0016] In addition, the formulations of the present invention may alsoinclude metal chelating agents, antioxidants or thiol-containingcompounds, such as ascorbic acid ester, monothioglycerol, cystein,tocopherols, butylated hydroxyanisole, sodium sulphate, sodiumbisulfite, and sodium metasulfite and preservatives such as phenol,chlorobutane, benzylalcohol, methyl parabens and propyl parabens. Theformulations of the present invention may also have an nitrogen blanketoverlay on the head space of the vial. Additionally, the formulations ofthe present invention may be include purging the formulation buffer withhelium, argon, or nitrogen.

BRIEF DESCRIPTION OF THE FIGURES

[0017] FIGS. 1A-1C illustrate the cDNA and corresponding deduced aminoacid sequence of KGF-2. The initial 35 or 36 amino acid residuesrepresent the putative leader sequence (underlined). The standard oneletter abbreviations for amino acids are used. Sequencing inaccuraciesare a common problem when attempting to determine polynucleotidesequences. Sequencing was performed using a 373 Automated DNA sequencer(Applied Biosystems, Inc.). Sequencing accuracy is predicted to begreater than 97% accurate. (SEQ ID NOs:1 and 2)

[0018] FIGS. 2(A)-2(C) depict stimulation of normal primary epidermalkeratinocyte proliferation by KGF-2 polypeptides of the invention.

[0019]FIG. 2(A) shows stimulation of normal primary epidermalkeratinocyte proliferation by KGF-2.

[0020]FIG. 2(B) shows the stimulation of normal primary epidermalkeratinocyte proliferation by KGF-2 Δ33.

[0021]FIG. 2(C) shows the stimulation of normal primary epidermalkeratinocyte proliferation by KGF-2 Δ28.

[0022]FIG. 3 shows bioactivity results for KGF-2 Δ33 liquid formulation,10 month stability.

[0023]FIG. 4 shows bioactivity results for KGF-2 Δ33 lyophilizedpreparation, 9 month stability.

[0024]FIG. 5 shows the effect of monothiolglycerol on KGF-2 bioactivity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] KGF-2 stimulates the proliferation of epidermal keratinocyes butnot mesenchymal cells such as fibroblasts. Thus, “a polypeptide havingKGF-2 protein-like activity” includes polypeptides that exhibit theKGF-2 activity, in the keratinocyte proliferation assay set forth belowand bind to FGF receptor isoforms 1-iiib and 2-iiib.

[0026] The present invention is directed to pharmaceutical andveterinary formulations of KGF-2 polypeptides. The KGF-2 polypeptidesare defined herein by reference to the polypeptide of FIG. 1 (SEQ IDNO:2) or that encoded by the deposited cDNA, and include fragments,derivatives and analogs of the polypeptide of FIG. 1 (SEQ ID NO:2) orthat encoded by the deposited cDNA which retain essentially the samebiological function as the parent polypeptide. The polypeptides employedin the present invention may be recombinant polypeptides, naturalpolypeptides or synthetic polypeptides, preferably recombinantpolypeptides.

[0027] It has been discovered that KGF-2 polypeptides exhibit pooractivity and stability at a pH of 4.5 or less, or at a pH above about8.0. The present inventors have discovered that KGF-2 polypeptidesoxidize and precipitate. These polypeptides present a difficultchallenge when attempting to formulate them for therapeutic purposes. Inorder to maintain physico-chemical properties and biological activity,KGF-2 polypeptides can be formulated with antioxidants, such as oxygenscavenging compounds, and/or a protein stabilizer, such as athiol-containing compound, and/or a metal-chelating agent, such as EDTA.Stabilization, as used herein, refers to the maintenance of bothphysico-chemical properties and substantial biological activity of theKGF-2 polypeptides over a given time period.

[0028] The formulations according to the present invention include gel,thickened solution, solution and lyophilized forms. Formulations arealso referred to herein as “pharmaceutical compositions” or“compositions.”

[0029] Injectable Formulations

[0030] Liquid Formulations

[0031] A first aspect of the present invention is directed to liquidformulations of KGF-2 polypeptides that comprise: a KGF-2 polypeptideand a buffer having a buffering capacity of between about pH 5.0 andabout pH 8.0, more preferably pH 5.5 to pH 6.5, most preferably pH 6.2.Useful buffers include buffers derived from phosphate, acetic, aconitic,citric, glutaric, malic, succinic and carbonic acids. Typically employedis an alkali or alkaline earth salt of one of the aformentioned acids.More preferably the buffer will be acetate or citrate, most preferablycitrate. For example, the formulation may comprise a composition formedby mixing a buffering amount of citric acid or a pharmaceuticallyacceptable salt thereof with KGF-2 Δ33 in water. The formulationalternatively may comprise a composition formed by mixing a bufferingamount of acetic acid or a pharmaceutically acceptable salt thereof withKGF-2 Δ33 in water. Preferable buffer concentrations are from about 5 mMto about 50 mM. Most preferably the acetate buffer will have aconcentration of about 20 mM and the citrate buffer will be about 10 mMto about 20 mM. The formulation may also include NaCl as a tonicifier ata concentration of from about 0.01 mM to about 150 mM, most preferablyat about 125 mM and a metal chelating agent, such as EDTA, at aconcentration of from about 0.1 mM to about 10 mM, most preferably atabout 1 mM Additionally, a liquid formulation of the present inventionmay also include one or more of (a) a stabilizing amount of anantioxidant, such as ascorbate and/or (b) a protein stabilizing amount athiol-compound, for example monothioglycerol (MTG). Without wishing tobe bound by theory, it is believed that thiol compounds such as MTGserve to protect free sulfhydryl groups present in the KGF-polypeptides.The storage conditions for the liquid formulation are typically at about2° C. to about 8° C. Alternatively, storage conditions are at or below−20° C. Most preferably, storage conditions are at about −20° C.Maintaining a KGF-2 liquid formulation in a frozen state limits theamount of oxidation to the polypeptide which in turn results in a stablepolypeptide formulation.

[0032] Preferably, a liquid formulation comprises:

[0033] (1) a therapeutically-effective amount of a KGF-2 polypeptide;

[0034] (2) an effective amount of a buffer having a buffering capacityof between about pH 5.0 and about pH 8.0; and

[0035] (3) a pharmaceutically acceptable diluent; and

[0036] (4) optionally one or more of the following:

[0037] (a) NaCl as a tonicifier,

[0038] (b) a chelating agent,

[0039] (c) a stabilizing amount of an antioxidant, and

[0040] (d) a stabilizing amount of a protein stabilizer.

[0041] The KGF-2 polypeptide preferably is maintained in solution.

[0042] Compositions of the present invention are manufactured byadmixing the above listed ingredients together, preferably inconcentrations and ratios as expressed herein.

[0043] Antioxidants that can be used in the liquid formulation includeascorbic acid, tocopherols, and butylated hydroxyanisole. In addition,stabilizers that can be used in the liquid formulation also includethiols such as cysteine, methionine and thioglycerols. Chelating agentsthat can be employed include ethylenediamine tetraacetic acid (EDTA), ordiethylenetriamine pentaacetic acid (DPTA), with EDTA being preferred.

[0044] Formulations of the present invention which include antioxidantsor thiols can increase the stability of the KGF-2 polypeptides. Thismakes it possible to have a pharmaceutical product with a longer shelflife.

[0045] More preferred liquid formulations comprise:

[0046] (1) a KGF-2 polypeptide in a concentration range of about 0.02 toabout 40 mg/ml (w/v), more preferably about 0.05 to about 30 mg/ml(w/v), even more preferably about 0.1 to about 20 mg/ml (w/v), stillmore preferably about 10 mg/ml (w/v), and most preferably about 0.2 to 4mg/ml;

[0047] (2) a buffer having a buffering capacity of between about pH 5.0and about pH 8.0 at a concentration range of about 5 mM to about 50 MM,preferably about 5 mM to about 30 mM; and

[0048] (3) a pharmaceutically acceptable diluent, preferably water, tobring the composition to a designated volume.

[0049] Useful buffers for the formulations of the present inventioninclude buffers derived from acetic, aconitic, citric, glutaric, malic,succinic, phosphate and carbonic acids. Typically employed is an alkalior alkaline earth salt of one of the aformentioned acids. Acetate andcitrate buffers, such as acetic acid or a pharmaceutically acceptablesalt thereof, or citric acid or a pharmaceutically acceptable saltthereof, are preferred. The preferable pH ranges for the solutionformulation is from about pH 5.0 to about pH 8.0, preferably pH 5.5 topH 6.5, and most preferably about pH 6.2. Sodium acetate or sodiumcitrate are the preferred buffering agents, with sodium citrate beingmost preferred.

[0050] To the above solution also preferably added are:

[0051] (4) a chelating agent, such as EDTA at a concentration range ofabout 0.1 mM to about 10 mM, more preferably at about 1 mM;

[0052] (5) NaCl at a concentration range of about 0.01 mM to about 150mM and more preferably at about 125 mM.

[0053] Optionally, a liquid formulation may also include a proteinstabilizing amount of a compound selected from the group consisting of:

[0054] (a) about 0.5% to about 2% w/v glycerol,

[0055] (b) about 0.1% to about 1% w/v methionine, or

[0056] (c) about 0.1% to about 2% w/v monothioglycerol.

[0057] Preferred embodiments of this aspect of the present inventioninclude a composition formed by mixing:

[0058] (1) a KGF-2 polypeptide in a concentration of about 0.02 to about40 mg/ml (w/v), more preferably about 0.1 to about 20 mg/ml, and mostpreferably about 0.2 to 4 mg/ml.

[0059] (2) 10 mM sodium citrate or 20 mM sodium acetate;

[0060] (3) 125 mM NaCl;

[0061] (4) 1 mM EDTA; and

[0062] (5) water as diluent.

[0063] More preferably, the solution formulation comprises a compositionformed by mixing:

[0064] (1) about 0.2 to about 4 mg/ml of a KGF-2 polypeptide;

[0065] (2) 20 mM sodium acetate;

[0066] (3) 125 mM NaCl;

[0067] (4) 1 mM EDTA; and

[0068] (5) water as a diluent,

[0069] wherein the solution is at about pH 6.2 and is stored at about−20° C.

[0070] The present inventors have discovered that KGF-2 polypeptidesreadily oxidize, aggregate and precipitate out of solution. Althoughoxidation of KGF-2 does not destroy biological activity, limiting theextent of oxidation of the product leads to a more stable product. Theinventors observed that if the liquid formulation is at a pH too low theKGF-2 polypeptide will lose biological activity. Additionally, as the pHof the solution approaches the pI for KGF-2, the protein willprecipitate out of solution. Thus, the inventors have determined thatliquid formulations should be maintained in the range of about pH 6.0 toabout pH 7.0, and that a pH of about 6.2 is most optimal for stabilizingthe KGF-2 polypeptide. Moreover, the inventors surprisingly determinedthat a citrate buffer specifically stabilizes the KGF-2 polypeptides.

[0071] Although, the use of a citrate buffer having at about pH 6.0-6.2provides a liquid formulation that reduces aggregation of the KGF-2polypeptide and increases stability, the liquid polypeptide formulationmay still be subject to oxidation and precipitation of KGF-2polypeptides. Thus, the inventors developed a lyophilized formulation asset forth below.

[0072] Lyophilized Formulations

[0073] A second aspect of the present invention is directed tolyophilizated formulations of KGF-2 polypeptides that comprise: a KGF-2polypeptide and a buffer having a buffering capacity of between about pH5.0 and about pH 8.0, more preferably pH 5.5 to pH 6.5, most preferablypH 6.2. Useful buffers include buffers derived from phosphate, aconitic,citric, glutaric, malic, succinic and carbonic acids. Typically employedis an alkali or alkaline earth salt of one of the aforementioned acids.More preferably the buffer will be phosphate or citrate, most preferablycitrate. For example, the formulation may comprise a composition formedby mixing a buffering amount of citric acid or a pharmaceuticallyacceptable salt thereof with KGF-2 Δ33 in water. The preferable bufferconcentration is from about 5 mM to about 50 mM and more preferably atabout 10 mM. Most preferably, the citrate buffer will be added in aconcentration of about 10 mM. Also preferably included in theformulation is NaCl as a tonicifier at a concentration of from about0.01 mM to about 150 MM, most preferably at about 20 mM and a metalchelating agent, such as EDTA, at a concentration of from about 0.01 mMto about 10 mM, most preferably at about 1 mM. In addition, bulkingagents/cryoprotectants such as sucrose, glycine, mannitol, trehalose orother pharmaceutically acceptable bulking agents are included in theformulation. The amount of bulking agent used will be such that thesolution is isotonic and is in a range of about 2% to about 10% w/v.Preferred concentrations are as follows: 5% mannitol, 7% sucrose, 8%trehalose, or 2% glycine+0.5% sucrose. More preferably, sucrose orsucrose/glycine mixture is used. Additionally, a lyophilized formulationof the present invention may also include one or more of (a) astabilizing amount of an antioxidant, such as ascorbate or (b) astabilizing amount of thiol-compound, for example monothioglycerol.Storage conditions for the lyophilized formulation are typically atabout 2° C. to about 25° C. More preferably storage conditions are at orbelow about 2° C. to about 8° C.

[0074] KGF-2 polypeptides are lyophilized at a concentration of about0.02 mg/ml to about 10 mg/ml of protein in the initial solution.

[0075] The initial lyophilization solution preferably comprises (inaddition to the KGF-2 polypeptides):

[0076] (1) an effective amount of citric acid or a pharmaceuticallyacceptable salt thereof, preferably sodium citrate, at a concentrationrange of about 5 mM to about 20 mM;

[0077] (2) NaCl at a concentration range of about 0.01 mM to about 125mM,

[0078] (3) EDTA at a concentration range of about 0.1 mM to about 10 mM

[0079] (4) one or more of sucrose, mannitol, glycine or trehalose at aconcentration range of about 2% w/v to about 15% w/v; and

[0080] (5) water.

[0081] The preferred pH range for the lyophilization buffer is fromabout 5.5 to about 8.0, preferably about pH 6.2.

[0082] More preferably, the lyophilization buffer comprises 10 mM sodiumcitrate, 20 mM sodium chloride, 1 mM disodium EDTA at pH 6.2 and 7%sucrose.

[0083] The lyophilized KGF-2 polypeptide formulations are reconstitutedin sterile water so as to maintain isotonic conditions of about 290mOsm. The KGF-2 polypeptides can be reconstituted in sterile water,optionally containing a stabilizing amount of antioxidants comprising:a) about 0.01% to about 2% w/v monothioglycerol, b) about 0.01% to about2% w/v ascorbic acid, c) about 0.01% to about 2% w/v methionine or d)combinations thereof.

[0084] The present invention includes lyophilization cycles that yield astable KGF-2 polypeptide formulation. The lyophilization cycle isdesigned to keep the KGF-2 polypeptide product below its collapsetemperature during the primary drying phase. Additionally, the moisturecontent is targeted to be preferably less than 5%, and more preferablyless than 2%. Such a protocol must be determined for any particularprotein on an individual basis. An example lyophilization cycle for theKGF-2 sucrose containing lyophilization formulation according to thepresent invention was determined to be as follows: Temperature (° C.)Pressure (mTorr) Time (min.) 5 (hold) atmospheric 60 5 to −45 (ramp)atmospheric 120 −45 (hold) atmospheric 120 −45 (hold) 75 to 100 60 −45to −20 (ramp) 75 to 100 125 −20 (hold) 75 to 100 2100 −20 to +25 (ramp)75 to 100 225 +25 (hold) 75 to 100 1020

[0085] The lyophilization formulation of the present invention providesa product with unexpectedly increased stability. Indeed, lyophilizedKGF-2 formulations of the present invention are biologically stable forat least 9 months at temperatures of up to 45° C. (FIG. 4).Reverse-phase HPLC demonstrated that the lyophilized KGF-2 formulationsof the present invention retained its physiochemical properties for upto 8 months at temperatures of at or below 45° C. and 75% relativehumidity. Stability for this length of time at such high temperatures isvery unusual for proteins.

[0086] Thickened and Gel Formulations

[0087] A third aspect of the invention is directed to thickened or gelformulations for KGF-2 polypeptides.

[0088] 1) Thickening Agents

[0089] Thickening agents may be added to the above described liquidformulations to increase the viscosity of the resulting formulation. Aformulation having an increased viscosity may be beneficial for topicalapplications where controlled release, adhering to the shape of a woundor avoiding run-off may be important. Such thickened formulations areemployed for topical uses such as wound healing, to treat skin disordersor any other use which could be treated via topical application of aKGF-2 pharmaceutical composition.

[0090] The thickening agent should raise the viscosity to about 50 toabout 10,000 cps, more preferably about 50 to about 1,000 cps and mostpreferably about 200 to about 300 cps. Viscosity is measured using arotating spindle viscometer. The most preferred concentration ofthickening agent is 0 to 5% (w/w). The thickened solution will stayliquid at all times.

[0091] Examples of appropriate thickening agents include, but are notlimited to water soluble etherified celluloses and carbomer (highmolecular weight polymers of acrylic acid cross-linked with either allylsucrose or allyl ethers of pentaerythritol). Examples of etherifiedcellulose are well known in the art (listed in USP) and include alkylcelluloses, hydroxyalkyl celluloses and alkylhydroxyalkyl cellulosese.g., methylcellulose, hydroxyethyl cellulose, hydroxy propyl cellulose,hydroxy propyl methylcellulose, and the like. In a further embodiment,the topical or incisional gel may comprise about 0 to about 20% byweight of a cellulose derivative having a molecular weight of about50,000 to about 700,000. In a preferred embodiment the cellulosederivative is present at about 2% to about 8% by weight and has amolecular weight in the range of about 80,000 to about 240,000.Preferred cellulose derivatives are hydroxypropylmethyl cellulose,methyl cellulose, carboxymethyl cellulose, and hydroxyethyl cellulose.

[0092] When thickening agents are added to the injectable formulations,detailed above, salts and buffering agents may be added or removed fromthe formulation for optimal stability. For example, the citrateconcentration may be increased. Preferred concentrations for citrate arefor example, about 10 mM to about 500 mM citrate, more preferably about10 mM to about 50 mM citrate and most preferably about 10 mM to about 20mM citrate. Additionally, the amount of sucrose may be decreased in thelyophilization formulation to a range from about 0% to about 5% sucrose.

[0093] Thickening agents may be added directly to a liquid formulationaccording to the present invention and then lyophilized. Alternatively,a lyophilized formulation according to the present invention may bereconstituted by adding a suitable diluent, most preferably water havinga thickening agent dissolved therein. Such thickened formulations couldbe administered by spray.

[0094] An example of a preferred thickened KGF-2 polypeptide solutionaccording to the present invention comprises a product formed by mixing:

[0095] (1) a topically effective amount of a KGF polypeptide, preferablyKGF-2 Δ33;

[0096] (2) about 10 mM to about 500 mM sodium citrate buffer;

[0097] (3) about 0.01 to about 150 mM NaCl;

[0098] (4) about 0.75 to about 1.27 mM, preferably about 1 mM EDTA;

[0099] (5) about 0.1% to about 7% sucrose;

[0100] (6) about 0.75 to about 1.5% (w/w) carboxy methyl cellulose orabout 0.5 to about 1.5% hydroxy propyl methyl cellulose or about 0.25 toabout 0.75% hydroxy ethyl cellulose or about 0 to 1% carbomer or anycombination thereof.

[0101] The a pH of such a formulation is most preferably pH 6.2.

[0102] 2) Gelling Agents

[0103] Another aspect of the present invention is directed to gelformulations for KGF-2 polypeptides. Gelling agents may be added toinjectable formulations of the present invention to provide aformulation that remains liquid at room temperature and solidifies whenapplied to the surface of the skin (at about 37° C.). Such formulationsmay be useful for topical applications where controlled release,adhearing to the shape of a wound or avoiding run-off may be important.Such gel formulations are employed for topical uses such as woundhealing, to treat skin disorders or any other use which could be treatedvia topical application of KGF-2 pharmaceutical composition.

[0104] Gel formulations for KGF-2 polypeptides according to the presentinvention comprise:

[0105] (1) a topically effective amount of a KGF polypeptide;

[0106] (2) a buffer;

[0107] (3) a pharmaceutically acceptable diluent, preferably water; and

[0108] (4) a gel-forming high molecular weight compound.

[0109] Viscosity of gel formulations of the present invention may be ina range of about 1 to about 10,000 cps at room temperature, mostpreferred about 20 to about 100 cps at room temperature. Viscosity ismeasured using a rotating spindle viscometer.

[0110] Gel forming high molecular weight compounds employed in thepresent invention are typically water-soluble polymers capable offorming a viscous aqueous solution, or non-water soluble,water-swellable polymers (e.g., collagen) that can also form a viscoussolution and that gel upon contact with skin.

[0111] Useful gel forming high molecular weight compounds may beselected from vinyl polymers, polyoxyethylene-polyoxypropylenecopolymers, polysaccharides, proteins, poly(ethylene oxide), acrylamidepolymers and derivatives and or salts thereof. Other compounds that canbe used to make pharmaceutical gel formulations used in healing woundscan be found in U.S. Pat. No. 5,427,778, which is herein fullyincorporated by reference.

[0112] Useful vinyl polymers (or substituted polyethylenes) includepolyacrylic acid, polymethacrylic acid, polyvinyl pyrrolidone andpolyvinyl alcohol. Useful polysaccharides include cellulose derivatives,glycosaminoglycans, agar, pectin, alginic acid, dextran, starch(α-amylose or amylopectin), and chitosan. Useful glycosaminoglycansinclude hyaluronic acid, chondroitin, chondroitin-4-sulfate, heparansulfate and heparin. The glycosaminoglycans may be used to enhance woundhealing in combination with any other gel forming polymer such as, forexample, collagen, gelatin, fibronectin. The acrylamide polymers may bepolyacrylamide or polymethacrylamide polymers.

[0113] Preferred high molecular weight gel forming compounds arepolyoxyethylene-polyoxypropylene block copolymers, especially thoseblock copolymers that are designated in the trade as PLURONICS (BASF) orPOLAXAMERS (BASF).

[0114] In one preferred embodiment, the gel of the present invention maycomprise about 10 to about 60% by weight of apolyoxyethylene-polyoxypropylene block copolymer having an averagemolecular weight of about 500 to 50,000. In a more preferred embodiment,the gel of the present invention may comprise about 14 to about 18% byweight of block copolymers having a molecular weight in the range 1,000to 15,000. Preferred block copolymers of the present invention arePluronic F108 and Pluronic F127.

[0115] Polyoxyethylene-polyoxpropylene block copolymers (Pluronic orPoloxamer) have great potential for use in topical drug delivery systemsbecause they exhibit reverse thermal gelation behavior, have good drugrelease characteristics as well as low toxicity. Gels are formed as thesolution is warmed. Thus, the gel is a low viscosity aqueous solution atroom temperature but when it contacts the mammalian body and is warmedby body temperature the viscosity increases as the solution gels.Pluronic gels can be used for the controlled delivery of KGF-2polypeptides to, for example, wounds and other such sites where topicaldelivery is desirable. KGF-2 polypeptides can be combined with thePluronic in the liquid state and applied to the wound. Gelation occursand effectively reduces the rate that the polypeptides are released tothe wound and thereby permits prolonged contact between the polypeptidesand the wound site. The benefits of using such gel formulations includekeeping the wound moist and having a pharmaceutical compound that isform-fitting to the wound or other such site where the compound may beapplied.

[0116] The preferred gel formulations for KGF-2 polypeptides accordingto the present invention comprises citrate buffer and a Pluronic. Theformulation may comprise an amount of citric acid or a pharmaceuticallyacceptable salt, thereof.

[0117] The gel formulation according to the present invention may alsoinclude an chelating agent, a stabilizing amount of antioxidants orthiols. The gel formulation will include a high molecular weightcompound, such as a Pluronic, or water-soluble etherified cellulose, andthe like in an amount that will form a gel. In the gel formulationaccording to the present invention, the KGF-2 polypeptides arepreferably in a concentration of about 0.01 mg/ml to about 10 mg/ml.

[0118] Preferably, the gel formulations are formed by mixing:

[0119] (1) a KGF-2 polypeptide, preferably KGF-2 Δ33, in a finalcalculated concentration of 0.01 mg/ml to about 10 mg/ml;

[0120] (2) an effective amount of a buffering agent;

[0121] (3) about 10% to about 60%, or more preferably about 14% to about18% by weight of a polyoxyethylene-polyoxypropylene block copolymerhaving an average molecular weight of about 500 to 50,000; and

[0122] (4) a pharmaceutically acceptable diluent, preferably water.

[0123] Another preferred gel formulation comprises:

[0124] (1) a pharmaceutically active amount of KGF-2 polypeptide;

[0125] (2) about 10 mM to about 500 mM sodium citrate;

[0126] (3) about 0.01 mM to about 150 mM NaCl;

[0127] (4) about 1 mM EDTA;

[0128] (5) about 0.1% to about 7% sucrose;

[0129] (6) about 14% to about 18% Pluronic F127; and

[0130] (7) water,

[0131] wherein the formulation is at a pH of about pH 6.2.

[0132] Most preferably, the gel formulation comprises:

[0133] (1) a KGF-2 polypeptide, preferably KGF-2 Δ33, at a concentrationrange of about 0.01 mg/ml to about 10 mg/ml (w/v), more preferably about0.1 mg/ml to about 3 mg/ml, and most preferably about 0.2 mg/ml;

[0134] (2) sodium citrate at a concentration range of about 5 mM toabout 20 mM;

[0135] (3) about 10% to about 25% (w/v), preferably about 15 to about25, and most preferably about 16% of Pluronic 127 or Poloxamer 407;

[0136] (4) about 6.7% to about 7.3% sucrose, preferably about 7%sucrose; and

[0137] (5) water to volume.

[0138] The gel formulation optionally further includes one or more ofthe following:

[0139] (6) EDTA at a concentration range of about 0.1 mM to about 10 mM.

[0140] (7) NaCl at a concentration range of about 0.01 mM to about 125mM. The preferred pH ranges for the gel formulation is from about pH 5.0to about pH 8.0, preferably pH 6.2 and the resulting gel formulationshould be isotonic.

[0141] 3) Additional Stabilizing Agents

[0142] All of the foregoing formulations of the present invention maybenefit from from anti-oxidants, metal chelating agents, thiolcontaining compounds and other general stabilizing agents. Examples ofsuch stabilizing agents, include, but are not limited to:

[0143] (a) about 0.5% to about 2% w/v glycerol,

[0144] (b) about 0.1% to about 1% w/v methionine,

[0145] (c) about 0.1% to about 2% w/v monothioglycerol,

[0146] (d) about 1 mM to about 10 mM EDTA,

[0147] (e) about 0.01% to about 2% w/v ascorbic acid,

[0148] (f) 0.003% to about 0.02% w/v polysorbate 80,

[0149] (g) 0.001% to about 0.05% w/v polysorbate 20,

[0150] (h) arginine,

[0151] (i) heparin,

[0152] (j) dextran sulfate,

[0153] (k) cyclodextrins, or

[0154] (l) combinations thereof.

[0155] Administration of KGF-2 Polypeptides

[0156] The KGF-2 polypeptide formulations of the present invention mayemploy suitable pharmaceutical diluents that are known to be useful inpharmaceutical compositions. Such a diluents include but are not limitedto, saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration. Preferably, the pharmaceutical compositions will beformulated according to the present invention, as indicated above. Wateris a preferred diluent.

[0157] The polypeptide having KGF-2 activity may be administered inpharmaceutical compositions in combination with one or morepharmaceutically acceptable excipients. It will be understood that, whenadministered to a human patient, the total daily usage of thepharmaceutical compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the type and degree ofthe response to be achieved; the specific composition, including whetheranother agent, if any, is employed; the age, body weight, generalhealth, sex and diet of the patient; the time of administration, routeof administration, and rate of excretion of the composition; theduration of the treatment; drugs (such as a chemotherapeutic agent) usedin combination or coincidental with the specific composition; and likefactors well known in the medical arts. Suitable formulations, known inthe art, can be found in Remington's Pharmaceutical Sciences (latestedition), Mack Publishing Company, Easton, Pa. The “effective amount” ofKGF-2 for purposes herein (including a KGF-2 effective amount) is thusdetermined by such considerations.

[0158] The pharmaceutical compositions of the present invention may beadministered in a convenient manner such as by the oral, rectal,topical, intravenous, intraperitoneal, intramuscular, intraarticular,subcutaneous, intranasal, inhalation, intraocular or intradermal routes.Parenteral and topical delivery are the preferred routes ofadministration. The term “parenteral” as used herein refers to modes ofadministration which include intravenous, intramuscular,intraperitoneal, intrasternal, subcutaneous and intraarticular injectionand infusion.

[0159] The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In most cases, the KGF-2 dosage is from about 1 μg/kg toabout 30 mg/kg body weight daily, taking into account the routes ofadministration, symptoms, etc. However, the dosage can be as low as0.001 μg/kg. For example, in the specific case of topical administrationdosages are preferably administered from about 0.01 μg to 9 mg per cm².In the case of intranasal and intraocular administration, dosages arepreferably administered from about 0.001 μg/ml to about 10 mg/ml, andmore preferably from about 0.05 mg/ml to about 4 mg/ml.

[0160] As a general proposition, the total pharmaceutically effectiveamount of the KGF-2 polypeptide administered parenterally will be in therange of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. If given continuously, the KGF-2 polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution or bottle solution may also be employed.

[0161] A course of KGF-2 polypeptide treatment to affect thefibrinolytic system appears to be optimal if continued longer than acertain minimum number of days, 7 days in the case of the mice. Thelength of treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

[0162] For parenteral administration, in one embodiment, the KGF-2polypeptide is formulated generally by mixing it at the desired degreeof purity, in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically acceptable carrier, i.e., one that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation. For example,the formulation preferably does not include oxidizing agents and othercompounds that are known to be deleterious to polypeptides.

[0163] Generally, the formulations are prepared by contacting the KGF-2polypeptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes. Suitable formulations, known in the art, can be found inRemington's Pharmaceutical Sciences (latest edition), Mack PublishingCompany, Easton, Pa.

[0164] KGF-2 polypeptides may also be administered to the eye to treatlacrimal gland injuries, disorders and pathologies in animals and humansas a liquid, drop, or thickend liquid, a gel.

[0165] KGF-2 polypeptides can also be intranasally administered to thenasal mucosa to treat disorders, injuries and pathologies of the nasalmucosa and sinus epithelia in animals and humans as liquid drops or in aspray form.

[0166] Generally, the formulations are prepared by contacting the KGF-2polypeptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes. Suitable formulations, known in the art, can be found inRemington 's Pharmaceutical Sciences (latest edition), Mack PublishingCompany, Easton, Pa.

[0167] The carrier may also contain minor amounts of suitable additivessuch as substances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0168] KGF-2 is typically formulated in such vehicles at a concentrationof about 0.01 μg/ml to 50 mg/ml, preferably 0.01 μg/ml to 10 mg/ml, at apH of about 5 to about 8, preferably about 6 to about 7, most preferablyabout pH 6.2. It will be understood that the use of certain of theforegoing excipients, carriers, or stabilizers will result in theformation of KGF-2 salts.

[0169] KGF-2 to be used for therapeutic administration may be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic KGF-2compositions may be placed into a container having a sterile accessport, for example, an intravenous solution bag or vial having a stopperpierceable by a hypodermic injection needle.

[0170] KGF-2 ordinarily will be stored in unit or multi-dose containers,for example, sealed ampules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 3-ml vials are filled with 1 ml ofsterile-filtered 1% (w/v) aqueous KGF-2 solution, and the resultingmixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized KGF-2 using Water-for-Injection which mayoptionally include one or more antioxidants.

[0171] Dosaging may also be arranged in a patient specific manner toprovide a predetermined concentration of an KGF-2 activity in the blood,as determined by an RIA technique, for instance. Thus patient dosagingmay be adjusted to achieve regular on-going trough blood levels, asmeasured by RIA, on the order of from 50 to 1000 ng/ml, preferably 150to 500 ng/ml.

[0172] The KGF-2 is also suitably administered by sustained-releasesystems. Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers 22:547-556(1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J.Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-release KGF-2compositions also include liposomally entrapped KGF-2. Liposomescontaining KGF-2 are prepared by methods known per se: DE 3,218,121;Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985); Hwanget al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal KGF-2 therapy.

[0173] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such containers can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides, agonists and antagonists of the presentinvention may be employed in conjunction with other therapeuticcompounds.

[0174] When the present inventors examined the biological activity andstability of the KGF-2 polypeptide prepared according to theformulations of the present invention, it was surprisingly discoveredthat the use of monothioglycerol may stabilize the KGF-2 polypeptidesand may behave as a potentiating agent for KGF-2 polypeptides in woundhealing. The optimal concentration range for the potentiating effect ofthe monothioglycerol was 0.1% to 2% w/v.

[0175] KGF-2 Polypeptides

[0176] KGF-2 stimulates the proliferation of epithelial cells andepidermal keratinocyes but not mesenchymal cells such as fibroblasts.Thus, “a polypeptide having KGF-2 protein-like activity” includespolypeptides that exhibit the KGF-2 activity, in the keratinocyteproliferation assay set forth below and U.S. application Ser. No.08/910,875 and can bind to the FGF receptor isoforms 1-iiib and 2-iiib.Although the degree of activity need not be identical to that of theKGF-2 protein, preferably, “a polypeptide having KGF-2 protein-likeactivity” exhibits substantially similar activity as compared to theKGF-2 protein (i.e., the candidate polypeptide exhibits greater activityor not more than tenfold less and, preferably, not more than abouttwofold less activity relative to the reference KGF-2 protein).

[0177] The KGF-2 polypeptides used in the formulations of the presentinvention may or may not have the N-terminal methionine, preferably thepolypeptide will be lacking the N-terminal methionine.

[0178] The KGF-2 cDNA clone was deposited as ATCC Deposit No. 75977 onDec. 16, 1994 at the American Type Culture Collection, PatentDepository, 10801 University Blvd, Manassas, Va. 20110-2209. Inaddition, a cDNA encoding KGF-2 Δ33 inserted into an expression vector,pHE4-5, was deposited at the ATCC on Jan. 9, 1998 as ATCC No. 209575.

[0179] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide, of FIG. 1 (SEQ ID NO:2) or that encoded by thedeposited cDNA, means a polypeptide which retains essentially the samebiological function or activity as such polypeptide. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

[0180] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

[0181] The fragment, derivative or analog of the polypeptide of FIG. 1(SEQ ID NO:2) or that encoded by the deposited cDNA may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

[0182] The terms “peptide” and “oligopeptide” are considered synonymous(as is commonly recognized) and each term can be used interchangeably asthe context requires to indicate a chain of at least to amino acidscoupled by peptidyl linkages. The word “polypeptide” is used herein forchains containing more than ten amino acid residues. All oligopeptideand polypeptide formulas or sequences herein are written from left toright and in the direction from amino terminus to carboxy terminus.

[0183] It will be recognized in the art that some amino acid sequencesof the KGF-2 polypeptide can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity. In general, it ispossible to replace residues which form the tertiary structure, providedthat residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein.

[0184] The polypeptides of the present invention are preferably in anisolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended are polypeptides thathave been purified, partially or substantially, from a recombinant hostcell or a native source.

[0185] The pharmaceutical formulations of the present invention includethe KGF-2 polypeptide of SEQ ID NO:2 (in particular the maturepolypeptide) and deletion mutants thereof, as well as polypeptides whichhave at least 90%, 95%, 96%, 97%, 98%, 99% similarity (more preferablyat least 90%, 95%, 96%, 97%, 98%, 99% identity) to the polypeptide ofSEQ ID NO:2 and deletion mutants thereof, and also include portions ofsuch polypeptides with such portion of the polypeptide (such as thedeletion mutants described below) generally containing at least 30 aminoacids and more preferably at least 50 amino acids.

[0186] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conservativeamino acid substituted sequence of one polypeptide to the sequence of asecond polypeptide.

[0187] By “% similarity” for two polypeptides is intended a similarityscore produced by comparing the amino acid sequences of the twopolypeptides using the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711) and the defaultsettings for determining similarity. Bestfit uses the local homologyalgorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) to find the best segment of similarity between twosequences.

[0188] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of a KGF-2polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the KGF-2 polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0189] As a practical matter, whether any particular polypeptide is atleast 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence shown in FIG. 1 [SEQ ID NO:2] or to the amino acidsequence encoded by deposited cDNA clone can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

[0190] KGF-2 Deletion Mutants

[0191] Native KGF-2 is relatively unstable in the aqueous state and itundergoes chemical and physical degradation resulting in loss ofbiological activity during processing and storage. Native KGF-2 is alsoprone to aggregation in aqueous solution, at elevated temperatures andit becomes inactivated under acidic conditions.

[0192] Particularly preferred KGF-2 polypeptides are the deletionmutants shown below (numbering starts with the first amino acid in theprotein (Met): Thr (residue 36)--Ser (residue 208) Ser (46)--Ser (208)Cys (37)--Ser (208) Pro (47)--Ser (208) Gln (38)--Ser (208) Glu(48)--Ser (208) Ala (39)--Ser (208) Ala (49)--(Ser (208) Leu (40)--Ser(208) Thr (50)--Ser (208) Gly (41)--Ser (208) Asn (51)--Ser (208) Gln(42)--Ser (208) Ser (52)--Ser (208) Asp (43)--Ser (208) Ser (53)--Ser(208) Met (44)--Ser (208) Ser (54)--Ser (208) Val (45)--Ser (208) Ser(55)--Ser (208) Ser (56)--Ser (208) Phe (57)--Ser (208) Ser (59)--Ser(208) Ser (62)--Ser (208) Ala (63)--Ser (208) Gly (64)--Ser (208) Arg(65)--Ser (208) Val (67)--Ser (208) Ser (69)--Ser (208) Val (77)--Ser(208) Arg (80)--Ser (208) Met (1), Thr (36), or Cys (37)--His (207) Met(1), Thr (36), or Cys (37)--Val (206) Met (1), Thr (36), or Cys(37)--Val (205) Met (1), Thr (36), or Cys (37)--Met (204) Met (1), Thr(36), or Cys (37)--Pro (203) Met (1), Thr (36), or Cys (37)--Leu (202)Met (1), Thr (36), or Cys (37)--Phe (201) Met (1), Thr (36), or Cys(37)--His (200) Met (1), Thr (36), or Cys (37)--Ala (199) Met (1), Thr(36), or Cys (37)--Ser (198) Met (1), Thr (36), or Cys (37)--Thr (197)Met (1), Thr (36), or Cys (37)--Asn (196) Met (1), Thr (36), or Cys(37)--Lys (195) Met (1), Thr (36), or Cys (37)--Arg (194) Met (1), Thr(36), or Cys (37)--Arg (193) Met (1), Thr (36), or Cys (37)--Thr (192)Met (1), Thr (36), or Cys (37)--Lys (191) Met (1), Thr (36), or Cys(37)--Arg (188) Met (1), Thr (36), or Cys (37)--Arg (187) Met (1), Thr(36), or Cys (37)--Lys (183)

[0193] Preferred embodiments include the N-terminal deletions Ala(63)-Ser (208) (KGF-2Δ28) and Ser (69)-Ser (208) (KGF-2Δ33). Otherpreferred N-terminal and C-terminal deletion mutants include: Ala(39)-Ser (208); Pro (47)-Ser (208); Val (77)-Ser (208); Glu (93)-Ser(208); Glu (104)-Ser (208); Val (123)-Ser (208); and Gly (138)-Ser(208). Other preferred C-terminal deletion mutants include: Met (1), Thr(36), or Cys (37)-Lys (153).

[0194] Also included by the present invention are deletion mutantshaving amino acids deleted from both the N-terminus and the C-terminus.Such mutants include all combinations of the N-terminal deletion mutantsand C-terminal deletion mutants described above, e.g., Ala (39)-His(200), Met (44)-Arg (193), Ala (63)-Lys (153), Ser (69)-Lys (153), etc.Those combinations can be made using recombinant techniques known tothose skilled in the art.

[0195] Thus, preferred KGF polypeptides for use in pharmaceuticalformulations of the present invention comprise N-terminal deletionmutants, including those comprising the amino acid sequence shown inFIG. 1 (SEQ ID NO:2) except for a deletion of at least the first 38N-terminal amino acid residues (i.e., a deletion of at least Met (1)-Gln(38)) but not more than the first 147 N-terminal amino acid residues ofFIG. 1 (SEQ ID NO:2). Alternatively, the formulation comprises a mutanthaving a deletion that will include at least the first 38 N-terminalamino acid residues (i.e., a deletion of at least Met (1)-Gln (38)) butnot more than the first 137 N-terminal amino acid residues of FIG. 1(SEQ ID NO:2). Alternatively, the formulation comprises a mutant havinga deletion that will include at least the first 46 N-terminal amino acidresidues but not more than the first 137 N-terminal amino acid residuesof FIG. 1 (SEQ ID NO:2). Alternatively, the formulation comprises amutant having a deletion that will include at least the first 62N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIG. 1 (SEQ ID NO:2). Alternatively,the formulation comprises a mutant having a deletion that will includeat least the first 68 N-terminal amino acid residues but not more thanthe first 137 N-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).Alternatively, the formulation comprises a mutant having a deletion thatwill include at least the first 76 N-terminal amino acid residues butnot more than the first 137 N-terminal amino acid residues of FIG. 1(SEQ ID NO:2). Alternatively, the formulation comprises a mutant havinga deletion that will include at least the first 92 N-terminal amino acidresidues but not more than the first 137 N-terminal amino acid residuesof FIG. 1 (SEQ ID NO:2). Alternatively, the formulation comprises amutant having a deletion that will include at least the first 103N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIG. 1 (SEQ ID NO:2). Alternatively,the formulation comprises a mutant having a deletion that will includeat least the first 122 N-terminal amino acid residues but not more thanthe first 137 N-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).

[0196] In addition to a formulation comprising a KGF-2 mutant with theranges of N-terminal deletion mutants described above, the presentinvention is also directed to a formulation having all combinations ofthe above described ranges, e.g., deletions of at least the first 62N-terminal amino acid residues but not more than the first 68 N-terminalamino acid residues of FIG. 1 (SEQ ID NO:2); deletions of at least thefirst 62 N-terminal amino acid residues but not more than the first 76N-terminal amino acid residues of FIG. 1 (SEQ ID NO:2); deletions of atleast the first 62 N-terminal amino acid residues but not more than thefirst 92 N-terminal amino acid residues of FIG. 1 (SEQ ID NO:2);deletions of at least the first 62 N-terminal amino acid residues butnot more than the first 103 N-terminal amino acid residues of FIG. 1(SEQ ID NO:2); deletions of at least the first 68 N-terminal amino acidresidues but not more than the first 76 N-terminal amino acid residuesof FIG. 1 (SEQ ID NO:2); deletions of at least the first 68 N-terminalamino acid residues but not more than the first 92 N-terminal amino acidresidues of FIG. 1 (SEQ ID NO:2); deletions of at least the first 68N-terminal amino acid residues but not more than the first 103N-terminal amino acid residues of FIG. 1 (SEQ ID NO:2); deletions of atleast the first 46 N-terminal amino acid residues but not more than thefirst 62 N-terminal amino acid residues of FIG. 1 (SEQ ID NO:2);deletions of at least the first 46 N-terminal amino acid residues butnot more than the first 68 N-terminal amino acid residues of FIG. 1 (SEQID NO:2); deletions of at least the first 46 N-terminal amino acidresidues but not more than the first 76 N-terminal amino acid residuesof FIG. 1 (SEQ ID NO:2); etc.

[0197] In another embodiment, formulations comprising C-terminaldeletion mutants are provided by the present invention. Preferably, theN-terminal amino acid residue of said C-terminal deletion mutants isamino acid residue 1 (Met), 36 (Thr), or 37 (Cys) of FIG. 1 (SEQ IDNO:2). Such formulations comprising mutants include those comprising theamino acid sequence shown in FIG. 1 (SEQ ID NO:2) except for a deletionof at least the last C-terminal amino acid residue (Ser (208)) but notmore than the last 55 C-terminal amino acid residues (i.e., a deletionof amino acid residues Glu (154)-Ser (208)) of FIG. 1 (SEQ ID NO:2).Alternatively, the formulation comprises a mutant having a deletion thatwill include at least the last C-terminal amino acid residue but notmore than the last 65 C-terminal amino acid residues of FIG. 1 (SEQ IDNO:2). Alternatively, the formulation comprises a mutant having adeletion that will include at least the last 10 C-terminal amino acidresidues but not more than the last 55 C-terminal amino acid residues ofFIG. 1 (SEQ ID NO:2). Alternatively, the formulation comprises a mutanthaving a deletion that will include at least the last 20 C-terminalamino acid residues but not more than the last 55 C-terminal amino acidresidues of FIG. 1 (SEQ ID NO:2). Alternatively, the formulationcomprises a mutant having a deletion that will include at least the last30 C-terminal amino acid residues but not more than the last 55C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2). Alternatively,the formulation comprises a mutant having a deletion that will includeat least the last 40 C-terminal amino acid residues but not more thanthe last 55 C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).Alternatively, the formulation comprises a mutant having a deletion thatwill include at least the last 50 C-terminal amino acid residues but notmore than the last 55 C-terminal amino acid residues of FIG. 1 (SEQ IDNO:2).

[0198] In addition to a formulation comprising a KGF-2 mutant with theranges of C-terminal deletion mutants described above, the presentinvention is also directed to a formulation having all combinations ofthe above described ranges, e.g., deletions of at least the lastC-terminal amino acid residue but not more than the last 10 C-terminalamino acid residues of FIG. 1 (SEQ ID NO:2); deletions of at least thelast C-terminal amino acid residue but not more than the last 20C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2); deletions of atleast the last C-terminal amino acid residue but not more than the last30 C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2); deletions ofat least the last C-terminal amino acid residue but not more than thelast 40 C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2);deletions of at least the last 10 C-terminal amino acid residues but notmore than the last 20 C-terminal amino acid residues of FIG. 1 (SEQ IDNO:2); deletions of at least the last 10 C-terminal amino acid residuesbut not more than the last 30 C-terminal amino acid residues of FIG. 1(SEQ ID NO:2); deletions of at least the last 10 C-terminal amino acidresidues but not more than the last 40 C-terminal amino acid residues ofFIG. 1 (SEQ ID NO:2); deletions of at least the last 20 C-terminal aminoacid residues but not more than the last 30 C-terminal amino acidresidues of FIG. 1 (SEQ ID NO:2); etc.

[0199] In yet another embodiment, the KGF-2 polypeptide can be adeletion mutant having amino acids deleted from both the N-terminal andC-terminal residues. Such mutants include all combinations of theN-terminal deletion mutants and C-terminal deletion mutants describedabove. Such mutants include those comprising the amino acid sequenceshown in FIG. 1 (SEQ ID NO:2) except for a deletion of at least thefirst 46 N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIG. 1 (SEQ ID NO:2) and a deletion ofat least the last C-terminal amino acid residue but not more than thelast 55 C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).Alternatively, a deletion can include at least the first 62, 68, 76, 92,103, or 122 N-terminal amino acids but not more than the first 137N-terminal amino acid residues of FIG. 1 (SEQ ID NO:2) and a deletion ofat least the last 10, 20, 30, 40, or 50 C-terminal amino acid residuesbut not more than the last 55 C-terminal amino acid residues of FIG. 1.Further included are all combinations of the above described ranges.

[0200] KGF-2 Substitution Mutants

[0201] Useful KGF-2 polypeptides include those having substitution ofamino acids. Native mature KGF-2 contains 44 charged residues, 32 ofwhich carry a positive charge. Depending on the location of suchresidues in the protein's three dimensional structure, substitution ofone or more of these clustered residues with amino acids carrying anegative charge or a neutral charge may alter the electrostaticinteractions of adjacent residues and may be useful to achieve increasedstability and reduced aggregation of the protein. Aggregation ofproteins cannot only result in a loss of activity but be problematicwhen preparing pharmaceutical formulations, because they can beimmunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967),Robbins et al., Diabetes 36: 838-845 (1987), Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10: 307-377 (1993)). Any modificationshould give consideration to minimizing charge repulsion in the tertiarystructure of the protein molecule. Thus, of special interest aresubstitutions of charged amino acid with another charge and with neutralor negatively charged amino acids. The latter results in proteins with areduced positive charge to improve the characteristics of KGF-2. Suchimprovements include increased stability and reduced aggregation of theanalog as compared to the native KGF-2 protein.

[0202] The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361: 266-268(1993), described certain TNF alpha mutations resulting in selectivebinding of TNF alpha to only one of the two known TNF receptors.

[0203] KGF-2 molecules may include one or more amino acid substitutions,deletions or additions, either from natural mutation or humanmanipulation. Examples of some preferred mutations are: Ala (49) Gln,Asn (51) Ala, Ser (54) Val, Ala (63) Pro, Gly (64) Glu, Val (67) Thr,Trp (79) Val, Arg (80) Lys, Lys (87) Arg, Tyr (88) Trp, Phe (89) Tyr,Lys (91) Arg, Ser (99) Lys, Lys (102) Gln, Lys 103(Glu), Glu (104) Met,Asn (105) Lys, Pro (107) Asn, Ser (109) Asn, Leu (111) Met, Thr (114)Arg, Glu (117) Ala, Val (120) Ile, Val (123) Ile, Ala (125) Gly, Ile(126) Val, Asn (127) Glu, Asn (127) Gln, Tyr (130) Phe, Met (134) Thr,Lys (136) Glu, Lys (137) Glu, Gly (142) Ala, Ser (143) Lys, Phe (146)Ser, Asn (148) Glu, Lys (151) Asn, Leu (152) Phe, Glu (154) Gly, Glu(154) Asp, Arg (155) Leu, Glu (157) Leu, Gly (160) His, Phe (167) Ala,Asn (168) Lys, Gln (170) Thr, Arg (174) Gly, Tyr (177) Phe, Gly (182)Gln, Ala (185) Val, Ala (185) Leu, Ala (185) Ile, Arg (187) Gln (190)Lys, Lys (195) Glu, Thr (197) Lys, Ser (198) Thr, Arg (194) Glu, Arg(194) Gln, Lys (191) Glu, Lys (191) Gln, Arg (188) Glu, Arg (188) Gln,Lys (183) Glu.

[0204] By the designation, for example, Ala (49) Gln is intended thatthe Ala at position 49 of FIG. 1 (SEQ ID NO:2) is replaced by Gln.

[0205] Changes are preferably of minor nature, such as conservativeamino acid substitutions that do not significantly affect the folding oractivity of the protein. Examples of conservative amino acidsubstitutions known to those skilled in the art are set forth below:Aromatic: phenylalanine tryptophan tyrosine Hydrophobic: leucineisoleucine valine Polar: glutamine asparagine Basic: arginine lysinehistidine Acidic: aspartic acid glutamic acid Small: alanine serinethreonine methionine glycine

[0206] Of course, the number of amino acid substitutions a skilledartisan would make depends on many factors, including those describedabove. Generally speaking, the number of substitutions for any givenKGF-2 polypeptide will not be more than 50, 40, 30, 20, 10, 5, or 3,depending on the objective. For example, a number of substitutions thatcan be made in the C-terminus of KGF-2 to improve stability.

[0207] Amino acids in KGF-2 that are essential for function can beidentified by methods well known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,Science 244:1081-1085 (1989). The latter procedure introduces singlealanine mutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity such as receptorbinding or in vitro and in vivo proliferative activity. (See, e.g.,Example 1). Sites that are critical for ligand-receptor binding can alsobe determined by structural analyzis such as crystalization, nuclearmagnetic resonance or photoaffinity labelling. (See for example: Smithet al., J. Mol. Biol., 224: 899-904 (1992); and de Vos et al. Science,255: 306-312 (1992).)

[0208] Other useful KGF polypeptides include polypeptides havingsubstitutions of serine for cysteine at amino acid positions 37 and 106and 150. An uneven number of cysteines means that at least one cysteineresidue is available for intermolecular crosslinks or bonds that cancause the protein to adopt an undesirable tertiary structure. NovelKGF-2 proteins that have one or more cysteines replaced by serine ore.g. alanine are generally purified at a higher yield of soluble,correctly folded protein. Although not wishing to be bound by theory, itis believed that the cysteine residue at position 106 is important forfunction. This cysteine residue is highly conserved among all other FGFfamily members.

[0209] Therapeutic Uses of KGF-2 Polypeptide Compositions

[0210] The polypeptides of the present invention may stimulatekeratinocyte cell growth and proliferation. Accordingly, compositions ofthe present invention can be employed to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of dermal wounds.These wounds may be of superficial nature or may be deep and involvedamage of the dermis and the epidermis of skin.

[0211] KGF-2 is useful for treating a number of diseases and conditions.For example, KGF-2 is active in vitro and in vivo in various woundhealing models. See, U.S. application Ser. Nos. 08/910,875, filed Aug.13, 1997 and Ser. No. 09/023,082 filed Feb. 13, 1998.

[0212] The individual to which KGF-2 is administered may heal wounds ata normal rate or may be healing impaired. When administered to anindividual who is not healing impaired, KGF-2 is administered toaccelerate the normal healing process. When administered to anindividual who is healing impaired, KGF-2 is administered to facilitatethe healing of wounds which would otherwise heal slowly or not at all. Anumber of afflictions and conditions can result in healing impairment.These afflictions and conditions include diabetes (e.g., Type IIdiabetes mellitus), treatment with both steroids and non-steroidpharmacological agents, and ischemic blockage or injury.

[0213] A number of growth factors have been shown to promote woundhealing in healing impaired individuals. These growth factors includegrowth hormone-releasing factor, platelet-derived growth factor, andbasic fibroblast growth factors. Thus, the present invention alsoencompasses the administration of KGF-2 compositions in conjunction withone or more additional growth factors or other agent which promoteswound healing.

[0214] The compositions of the present invention also promote thehealing of anastomotic and other wounds caused by surgical procedures inindividuals which both heal wounds at a normal rate and are healingimpaired.

[0215] The compositions of the present invention may also be employed tostimulate differentiation of cells, for example muscle cells, cellswhich make up nervous tissue, prostate cells, and lung cells.

[0216] The compositions of the present invention are clinically usefulin stimulating wound healing of wounds including surgical wounds,excisional wounds, deep wounds involving damage of the dermis andepidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds,diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venousstasis ulcers, and burns resulting from heat exposure to extremetemperatures of heat or cold, or exposure to chemicals, in normalindividuals and those subject to conditions which induce abnormal woundhealing such as uremia, malnutrition, vitamin deficiencies, obesity,infection, immunosuppression and complications associated with systemictreatment with steroids, radiation therapy, and antineoplastic drugs andantimetabolites. The compositions are also useful for promoting thehealing of wounds associated with ischemia and ischemic injury, e.g.,chronic venous leg ulcers caused by an impairment of venous circulatorysystem return and/or insufficiency; for promoting dermal reestablishmentsubsequent to dermal loss; increasing the tensile strength of epidermisand epidermal thickness; and increasing the adherence of skin grafts toa wound bed and to stimulate re-epithelialization from the wound bed.

[0217] Other therapeutic uses for the KGF-2 polypeptides include, butare not limited to, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of treating burnsand skin defects such as psoriasis and epidermolysis bullosa. KGF-2 canbe used to increase the adherence of skin grafts to a wound bed and tostimulate re-epithelialization from the wound bed. KGF-2 can also beused to reduce the side effects of gut toxicity that result fromradiation, chemotherapy treatments or viral infections. KGF-2 can beused to treat diseases and conditions of the liver, lung, kidney,breast, pancreas, stomach, small intestine, and large intestine. KGF-2can be used to treat inflamamatory bowel diseases, diabetes,thrombocytopenia, hypofibrinogenemia, hypoalbuminemia, hypoglobulinemia,hemorrhagic cystitis, xerostomia, keratoconjunctivitis sicca. KGF-2 canbe used to stimulate the epithelial cells of the salivary glands,lacrimal glands and stimulating re-epithelialization of the sinuses andthe growth of nasal mucosa.

[0218] A number of other indications that can be treated by thecomposition of the present invention are described in U.S. applicationSer. Nos. 08/910,875, and 09/023,082 and are herein incorporated byreference.

[0219] The present invention is directed to novel liquid and lyophilyzedformulations of KGF-2 and deletion mutants thereof. This inventionfurther relates to formulations of KGF-2 for therapeutic use. Theformulations provide superior stability to the active KGF-2 polypeptidesand in some instances, potentiate and dramatically increase thewound-healing activity of the polypeptides.

[0220] As used herein, by “individual” is intended an animal, preferablya mammal (such as apes, cows, horses, pigs, boars, sheep, rodents,goats, dogs, cats, chickens, monkeys, rabbits, ferrets, whales, anddolphins), and more preferably a human.

[0221] The KGF-2 Δ33 polypeptide used in the formulations of the presentinvention may or may not have the N-terminal methionine, preferably thepolypeptide will be lacking the N-terminal methionine. Stability of theKGF-2 polypeptide formulations of the present invention is determined byproliferation assays, as described herein below.

[0222] Keratinocyte Proliferation Assays

[0223] Dermal keratinocytes are cells in the epidermis of the skin. Thegrowth and spreading of keratinocytes in the skin is an importantprocess in wound healing. A proliferation assay of keratinocyte istherefore a valuable indicator of protein activities in stimulatingkeratinocyte growth and consequently, wound healing.

[0224] Keratinocytes are, however, difficult to grow in vitro. Fewkeratinocyte cell lines exist. These cell lines have different cellularand genetic defects. In order to avoid complications of this assay bycellular defects such as loss of key growth factor receptors ordependence of key growth factors for growth, primary dermalkeratinocytes are chosen for this assay. These primary keratinocytes areobtained from Clonetics, Inc. (San Diego, Calif.).

[0225] The bioactivity of KGF-2 polypeptides can be determined by a cellproliferation assay employing murine Baf3 2b cells that have beentransfected with the fibroblast growth factor 2iiib receptor(FGFR2iiib). Proliferation of the cells is measured by the incorporationof [Methyl-³H]-thymidine after the cells have been exposed to theprotein as described below. The assay is carried out in a 96 well tissueculture cluster plate with about 22,000 Baf3 2b cells in each well. Thecells are exposed to different concentrations of a KGF-2 polypeptide intriplicate and incubated at 37° C. in a CO₂ incubator for approximately48 hours. An approximate amount of cell media containing labelledthymidine is subsequently added into each well and the incubation iscontinued for another 5 hours. The cells are then harvested on a glassfiber filter mat, in the 96 well format, using a cell harvester. Thefilter mats are dried and radioactivity incorporated into each sample iscounted using a flat-bed liquid scintillation counter. Under these assayconditions, cells exposed to KGF-2 show an increased incorporation ofradioactivity compared to control cells that have been treated eitherwith an appropriate dilution of the placebo buffer or simply withphosphate buffered saline.

[0226] Another useful keratinocyte proliferation assay is with AlamarBlue. Alamar Blue is a viable blue dye that is metabolized by themitochondria when added to the culture media. The dye then turns red intissue culture supernatants. The amounts of the red dye may be directlyquantitated by reading difference in optical densities between 570 nmand 600 nm. This reading reflects cellular activities and cell number.

[0227] Normal primary dermal keratinocytes (CC-0255, NHEK-Neo pooled)are purchased from Clonetics, Inc. These cells are passage 2.Keratinocytes are grown in complete keratinocyte growth media (CC-3001,KGM; Clonetics, Inc.) until they reach 80% confluency. The cells aretrypsinized according to the manufacturer's specification. Briefly,cells are washed twice with Hank's balanced salt solution. 2-3 ml oftrypsin is added to cells for about 3-5 min at room temperature. Trypsinneutralization solution is added and cells are collected. Cells are spunat 600×g for 5 min at room temperature and plated into new flasks at3,000 cells per square centi-meter using pre-warmed media.

[0228] For the proliferation assay, plate 1,000-2,000 keratinocytes perwell of the Corning flat bottom 96-well plates in complete media exceptfor the outermost rows. Fill the outer wells with 200 μl of sterilewater. This helps to keep temperature and moisture fluctuations of thewells to the minimum. Grow cells overnight at 37° C. with 5% CO₂. Washcells twice with keratinocyte basal media (CC-3101, KBM, Clonetics,Inc.) and add 100 μl of KBM into each well. Incubate for 24 hours.Dilute growth factors in KBM in serial dilution and add 100 μl to eachwell. Use KGM as a positive control and KBM as a negative control. Sixwells are used for each concentration point. Incubate for two to threedays. At the end of incubation, wash cells once with KBM and add 100 μlof KBM with 10% v/v alamarBlue pre-mixed in the media. Incubate for 6 to16 hours until media color starts to turn red in the KGM positivecontrol. Measure O.D. 570 nm minus O.D. 600 nm by directly placingplates in the plate reader.

[0229] Construction of KGF-2 Deletion Mutants

[0230] Useful deletion mutants for use in compositions of the presentinvention can be constructed by the following protocol.

[0231] Deletion mutants were constructed from the 5′ terminus and 3′terminus of KGF-2 gene using an optimized KGF-2 construct as a template.The deletions were selected based on regions of the gene that mightnegatively affect expression in E. coli. For the 5′ deletion the primerslisted below were used as the 5′ primer. These primers contain theindicated restriction site and an ATG to code for the initiatormethionine. The KGF-2 (FGF-12) 208 amino acid 3′ HindIII primer was usedfor the 3′ primer. PCR amplification for 25 rounds was performed usingstandard conditions. The products for the KGF-236aa/208aa deletionmutant were restricted BspHI for the 5′ site and HindIII for the 3′ siteand cloned into the pQE60 which has bee digested with BspHI and HindIII.All other products were restricted with NcoI for the 5′ restrictionenzyme and HindIII for the 3′ site, and cloned into the pQE60 which hadbeen digested with NcoI and HindIII. For KGF-2 (FGF-12), 36aa/153aa and128aa 3′ HindIII was used as the 3′ primer with FGF-12 36aa/208aa as the5′ primer. For FGF-12 62aa/153aa, 128aa 3′ HindIII was used as the 3′primer with FGF-12 62aa/208aa as the 5′ primer. The nomenclature of theresulting clones indicates the first and last amino acid of thepolypeptide that results from the deletion. For example, KGF-236aa/153aa indicates that the first amino acid of the deletion mutant isamino acid 36 and the last amino acid is amino acid 153 of KGF-2. Theconstruction of these KGF-2 deletion mutants are also described in U.S.application Ser. Nos. 08/910,875, and 09/023,082 and are hereinincorporated by reference. Further, as indicated in below, each mutanthas N-terminal Met added thereto. However, the KGF-2 deletionpolypeptides used in the formulations according to the present inventionmay or may not have the N-terminal methionine, preferably thepolypeptide will be lacking the N-terminal methionine.

[0232] Sequences of the Deletion Primers

[0233] FGF12 36aa/208aa

[0234] 5′ BsphI GGACCCTCATGACCTGCCAGGCTCTGGGTCAGGAC (SEQ ID NO:3)

[0235] FGF12 63aa/208aa

[0236] 5′ NcoI GGACAGCCATGGCTGGTCGTCACGTTCG (SEQ ID NO:4)

[0237] FGF12 77aa/208aa

[0238] 5′ NcoI GGACAGCCATGGTTCGTTGGCGTAAACTG (SEQ ID NO:5)

[0239] FGF12 93aa/208aa

[0240] 5′ NcoI GGACAGCCATGGAAAAAACGGTAAAGTTTC (SEQ ID NO:6)

[0241] FGF12 104aa/208aa

[0242] 5′ NcoI GGACCCCCATGGAGAACTGCCCGTAGAGC (SEQ ID NO:7)

[0243] FGF12 123aa/208aa

[0244] 5′ NcoI GGACCCCCATGGTCAAAGCCATTAACAGCAAC (SEQ ID NO:8)

[0245] FGF12 138aa/208aa

[0246] 5′ NcoI GGACCCCCATGGGGAAACTCTATGGCTCAAAAG (SEQ ID NO:9)

[0247] FGF12 3′ HindIII: (Used for all above deletion clones)

[0248] CTGCCCAAGCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID NO:10)

[0249] FGF12 36aa/153aa

[0250] 5′ BsphI (as above)

[0251] 3′HindIII CTGCCCAAGCTTATTACTTCAGCTTACAGTCATTGT (SEQ ID NO:11)

[0252] FGF12 63aa/153aa

[0253] 5′NcoI and 3′HindIII, as above.

[0254] Construction of N-terminal Deletion Mutant KGF-2Δ33

[0255] Construction of KGF-2 Δ33 in pQE6

[0256] To permit Polymerase Chain Reaction directed amplification andsub-cloning of KGF2Δ33 into the E.coli protein expression vector, pQE6,two oligonucleotide primers (5952 and 19138) complementary to thedesired region of KGF2 were synthesized with the following basesequence.

[0257] Primer 5952: 5′ GCGGCACATGTCTTACAACCACCTGCAGGGTG3′ (SEQ ID NO:12)

[0258] Primer 19138: 5′ GGGCCCAAGCTTATGAGTGTACCACCAT 3′ (SEQ ID NO:13)

[0259] In the case of the N-terminal primer (5952), an AflIIIrestriction site was incorporated, while in the case of the C-terminalprimer (19138) a HindIII restriction site was incorporated. Primer 5952also contains an ATG sequence adjacent and in frame with the KGF2 codingregion to allow translation of the cloned fragment in E. coli, whileprimer 19138 contains two stop codons (preferentially utilized in E.coli) adjacent and in frame with the KGF2 coding region which ensurescorrect translational termination in E. coli.

[0260] The Polymerase Chain Reaction was performed using standardconditions well known to those skilled in the art and the nucleotidesequence for the mature KGF-2 (aa 36-208) as template. The resultingamplicon was restriction digested with AflIII and HindIII and subclonedinto NcoI/HindIII digested pQE6 protein expression vector.

[0261] Construction of KGF-2 Δ33 in pHE1

[0262] To permit Polymerase Chain Reaction directed amplification andsubcloning of KGF2Δ33 into the E.coli expression vector, pHE1, twooligonucleotide primers (6153 and 6150) corresponding to the desiredregion of KGF2 were synthesized with the following base sequence.

[0263] Primer 6153: 5′ CCGGCGGATCCCATATGTCTTACAACCACCTGCAGG3′ (SEQ IDNO:14)

[0264] Primer 6150: 5′ CCGGCGGTACCTTATTATGAGTGTACCACCATTGG3′ (SEQ IDNO:15)

[0265] In the case of the N-terminal primer (6153), an NdeI restrictionsite was incorporated, while in the case of the C-terminal primer (6150)an Asp718 restriction site was incorporated. Primer 6153 also containsan ATG sequence adjacent and in frame with the KGF2 coding region toallow translation of the cloned fragment in E. coli, while primer 6150contains two stop codons (preferentially utilized in E. coli) adjacentand in frame with the KGF2 coding region which ensures correcttranslational termination in E. coli.

[0266] The Polymerase Chain Reaction was performed using standardconditions well known to those skilled in the art and the nucleotidesequence for the mature KGF-2 (aa 36-208) as template. The resultingamplicon was restriction digested with NdeI and Asp718 and subclonedinto NdeI/Asp718 digested pHE1 protein expression vector.

[0267] Nucleotide Sequence of KGF-2 Δ33

[0268] ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTC CAATGGTGGTACACTCATAA (SEQID NO:16)

[0269] Amino Acid sequence of KGF-2 Δ33

[0270] MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMVVHS (SEQ ID NO:17)

[0271] B. Construction of an Optimized KGF-2 Δ33 Polynucleotide Sequence

[0272] In order to increase the expression levels of KGF2Δ33 in E.coli,the codons of the complete gene were optimized to match those mosthighly used in E.coli. As the template utilized to generate the KGF2Δ33was codon optimized within the N-terminal region, the C-terminal aminoacids (84-208) required optimization.

[0273] Firstly, amino acids 172-208 were codon optimized to generateKGF2Δ33(s172-208). This was achieved by an overlapping PCR strategy.Oligonucleotides PM07 and PM08 (corresponding to amino acids 172-208)were combined and annealed together by heating them to 70° C. andallowing them to cool to 37° C. The annealed oligonucleotides were thenutilized as template for a standard PCR reaction which was directed byprimers PM09 and PM10. In a separate PCR reaction following standardconditions well known to those skilled in the art and using KGF2Δ33 astemplate, oligonucleotides PM05 (which overlaps with the Pst1 sitewithin coding region of KGF2) and PM11 were used to amplify the regionof KGF2 corresponding to amino acids 84-172. In a third PCR reaction,the product of the first PCR reaction (corresponding to codon optimizedamino acids 172-208) and the product of the second PCR reaction(corresponding to codon non-optimized amino acids 84-172) were combinedand used as template for a standard PCR reaction directed byoligonucleotides PM05 and PM10. The resulting amplicon was digested withPst1/HindIII and sub-cloned into Pst1/HindIII digested pQE6KGF2Δ33,effectively substituting the corresponding non codon optimized region,and creating pQE6KGF2Δ33(s172-208).

[0274] To complete the codon optimization of KGF2, a synthetic genecodon optimized for the region of KGF2 corresponding to amino acids84-172 was generated utilizing overlapping oligonucleotides. Fouroligonucleotides (PM3 1, PM32, PM33 and PM 34) were combined and sevencycles of the following PCR was performed: 94° C., 30 secs; 46.5° C., 30secs; and 72° C., 30 secs.

[0275] A second PCR reaction directed by primers PM35 and PM36 was thenperformed following standard procedures, utilizing 1 μl of the first PCRreaction as template. The resulting codon optimized gene fragment wasthen digested with Pst1/Sal1 and subcloned into Pst1/Sal1 digestedpQE6KGF2Δ33(s172-208) to create a fully optimized KGF2 encoding gene,pQE6KGF2Δ33s.

[0276] To create an alternative E.coli protein expression vector,KGF2Δ33s was PCR amplified utilising primers PM102 and PM130 onpQE6KGF2Δ33s. The resulting amplicon was digested with NdeI and EcoRVand subcloned into the pHE1 expression vector which had been digestedwith NdeI and Asp718 (blunt ended) to create pHE1Δ33s.

[0277] Oligonucleotide Sequences used in construction of codon optimizedKGF-2 Δ33s:

[0278] PM05: CAACCACCTGCAGGGTGACG (SEQ ID NO:18)

[0279] PM07: AACGGTCGACAAATGTATGTGGCACTGAACGGTAAAGGTGCTCCACGTCGTGGTCAGAAAACCCGTCGTAAAAACACC (SEQ ID NO:19)

[0280] PM08: GGGCCCAAGCTTAAGAGTGTACCACCATTGGCAGAAAGTGAGCAGAGGTGTTTTTACGACGGGTTTTCTGACCACG (SEQ ID NO:20)

[0281] PM09: GCCACATACATTTGTCGACCGTT (SEQ ID NO:21)

[0282] PM10: GGGCCCAAGCTTAAGAGTG (SEQ ID NO:22)

[0283] PM11: GCCACATACATTTGTCGACCGTT (SEQ ID NO:23)

[0284] PM31: CTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCCTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGTACCAA G (SEQ ID NO:24)

[0285] PM32: AGCTTTAACAGCAACAACACCGATTTCAACGGAGGTGATTTCCAGGATGGAGTACGGGCAGTTTTCTTTCTTGGTACCAGAAACTTTAC C (SEQ ID NO:25)

[0286] PM33: GGTGTTGTTGCTGTTAAAGCTATCAACTCCAACTACTACCTGGCTATGAACAAGAAAGGTAAACTGTACGGTTCCAAAGAATTTAACAA C (SEQ ID NO:26)

[0287] PM34: GTCGACCGTTGTGCTGCCAGTTGAAGGAAGCGTAGGTGTTGTAACCGTTTTCTTCGATACGTTCTTTCAGTTTACAGTCGTTGTTAAATT CTTTGGAACC (SEQ IDNO:27)

[0288] PM35: GCGGCGTCGACCGTTGTGCTGCCAG (SEQ ID NO:28)

[0289] PM36: GCGGCCTGCAGGGTGACGTTCGTTGG (SEQ ID NO:29)

[0290] PM102: CCGGCGGATCCCATATGTCTTACAACCACCTGCAGG (SEQ ID NO:30)

[0291] PM130: CGCGCGATATCTTATTAAGAGTGTACCACCATTG (SEQ ID NO:31)

[0292] Nucleotide Sequence of KGF-2 Δ33(s172-208)

[0293] ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCCTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGTACCAAGAAAGAAAACTGCCCGTACTCCATCCTGGAAATCACCTCCGTTGAAATCGGTGTTGTTGCTGTTAAAGCTATCAACTCCAACTACTACCTGGCTATGAACAAGAAAGGTAAACTGTACGGTTCCAAAGAATTTAACAACGACTGTAAACTGAAAGAACGTATCGAAGAAAACGGTTACAACACCTACGCTTCCTTCAACTGGCAGCACAACGGTCGACAAATGTATGTGGCACTGAACGGTAAAGGTGCTCCACGTCGTGGTCAGAAAACCCGTCGTAAAAACACCTCTGCTC ACTTTCTGCCAATGGTGGTACACTCTTAA (SEQ IDNO:32)

[0294] Amino Acid Sequence of KGF-2 Δ33(s172-208)

[0295] MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMVVHS (SEQ ID NO:33)

EXAMPLES Example 1 KGF-2 Liquid Formulation

[0296] The following ingredients were mixed to create a liquid KGF-2 Δ33formulation is a liquid that is stored at −20° C.

[0297] 2 mg/ml KGF-2 Δ33 polypeptide,

[0298] 20 mM sodium acetate,

[0299] 125 mM sodium chloride,

[0300] 1 mM EDTA,

[0301] Water, pH 6.2.

[0302] This formulation retained its in vitro bioactivity for up to 10months at storage conditions at or below 2 to 8° C. The bioactivity at10 months is shown in FIG. 3. This formulation retained all itsphysico-chemical properties for up to 11 months at storage conditions ator below.

[0303] Bioactivity was measured using a cell proliferation assay asfollows. BaF3 cells, were routinely grown and maintained in RPMI 1640medium containing 10% NBCS, 10% WEHI cell conditioned medium, 2 mMglutamine, 600 μg/ml GENETICIN, 1 μl β mercaptoethanol/500 ml growthmedium, 50 units penicillin and 50 μg/ml streptomycin (Ornitz, D., M. etal (1996) J. Biol. Chem. 271:15292-15297). For cell proliferationassays, BaF3 cells were harvested by centrifugation and washed withBasal medium (this has the same composition as the growth medium, butcontains no WEHI conditioned medium and is supplemented with 1 μg/mlheparin). Following this operation the cells were resuspended in basalmedium and 22,000 cells/180 μl were plated/well in a 96 well cellculture cluster dish. Appropriate dilutions (10× higher than therequired final concentration) of KGF 2 were made in PBS in another 96well plate and added to the cells to a final volume of 200 μl. The cellplates were incubated in a 37° C., 5% CO₂ incubator for 36-40 hr. and0.5 μCi methyl-³H thymidine in 50 μl basal medium was added to eachwell. The plates were incubated for another 5 hr. in the incubator andcells were harvested by filtration on a glass fiber filter using aTomtec Harvester 96. Incorporated thymidine was counted on a Wallac βplate scintillation counter.

Example 2 KGF-2 Lyophilized Formulation

[0304] The following ingredients were mixed to create a KGF-2 Δ33lyophilized formulation.

[0305] 10 mg/ml KGF-2 Δ33,

[0306] 10 mM sodium citrate,

[0307] 20 mM sodium chloride,

[0308] 1 mM EDTA,

[0309] 7% w/v sucrose,

[0310] water (removed upon lyophiliztion)

[0311] pH 6.2.

[0312] This formulation retained its in vitro bioactivity for up to 9months at storage conditions at or below 45° C. The bioactivity at 9months is shown in FIG. 4. Bioactivity was measured using the cellproliferation assay detailed in Example 1. Reverse-phase HPLCdemonstrated that the formulation retained its physio-chemicalproperties for up to 8 months at temperatures of at or below 45° C. and75% relative humidity.

Example 3 KGF-2 in a Thickened Formulation

[0313] The following ingredients were mixed to create a KGF-2 Δ33thickened formulation.

[0314] 2 mg/ml KGF-2 Δ33,

[0315] 10 mM sodium citrate,

[0316] 20 mM sodium chloride,

[0317] 1 mM EDTA,

[0318] 7% w/v sucrose,

[0319] 1.25% carboxy methyl cellulose,

[0320] water

[0321] pH 6.2.

[0322] This formulation is prepared by adding KGF-2 Δ33 polypeptide tothe carboxy methyl cellulose solution. The viscosity of the resultingformulation was about 250 cps as determined by rotating spindleviscometer. The KGF-2 polypeptide retained bioactivity in the presenceof carboxy methyl cellulose. Bioactivity of the formulation was assayedusing the cell proliferation assay detailed in Example 1.

Example 4 KGF-2 in a Gel Formulation

[0323] The following ingredients were mixed to create a KGF-2 Δ33 gelformulation.

[0324] 2 mg/ml KGF-2 Δ33,

[0325] 10 mM sodium citrate,

[0326] 20 mM sodium chloride,

[0327] 1 mM EDTA,

[0328] 7% w/v sucrose,

[0329] 16% Pluronic F127,

[0330] water

[0331] pH 6.2.

[0332] KGF-2 Δ33 is added to a Pluronic solution at about 2° C. to about8° C. The viscosity of the resulting formulation was about 50 cps at 20°C. and solid at about 37° C. KGF-2 retained bioactivity in the presenceof Pluronic F127 as measured by the cell proliferation assay detailed inExample 1.

Example 5 Activation of KGF2 by Monothioglycerol

[0333] KGF-2 Δ33 protein stock formulations (0.1 to 2.0 mg/ml) wereprepared with or without monothioglycerol (MTG).

[0334] The protein formulations were diluted in 1× phosphate bufferedsaline (PBS) at pH 7.2 to attain the required concentrations for use inthe cell proliferation assays.

[0335] Cell Culture

[0336] BaF32b cells, were routinely grown and maintained in RPMI 1640medium containing 10% NBCS, 10% WEHI cell conditioned medium, 2 mMglutamine, 600 μg/ml GENETICIN, 1 μl β mercaptoethanol/500 ml growthmedium, 50 units penicillin and 50 μg/ml streptomycin (Ornitz, D., M. etal (1996) J. Biol. Chem. 271:15292-15297).

[0337] Cell Proliferation Assays

[0338] For cell proliferation assays, BaF32b cells were harvested bycentrifugation and washed with Basal medium (this has the samecomposition as the growth medium, but contains no WEHI conditionedmedium and is supplemented with 1 μg/ml heparin). Following thisoperation the cells were resuspended in basal medium and 22,000cells/180 μl were plated/well in a 96 well cell culture cluster dish.Appropriate dilutions (10× higher than the required final concentration)of KGF 2 were made in PBS in another 96 well plate and added to thecells to a final volume of 200 μl. The cell plates were incubated in a37° C., 5% CO₂ incubator for 36-40 hr. and 0.5 μCi methyl-³H thymidinein 50 μl basal medium was added to each well. The plates were incubatedfor another 5 hr. in the incubator and cells were harvested byfiltration on a glass fiber filter using a Tomtec Harvester 96.Incorporated thymidine was counted on a Wallac β plate scintillationcounter.

[0339] Results

[0340] A. Effect of MTG Concentration on KGF-2 Activity

[0341] The cell proliferation assay was carried out with KGF-2 exposedto different concentrations of monothioglycerol (MTG). Control samplescontained no excipient. With MTG, stimulation of KGF-2 activity wasobserved with various concentrations of MTG is shown in FIG. 5. Theincrease in activity was between 10-150% of control depending on theconcentration of MTG used. This enhancement of cell proliferationactivity was not observed with other members of this growth factorfamily. From these observations, it was concluded that stimulation ofKGF-2 activity by MTG was quite specific.

[0342] Conclusions

[0343] Monothioglycerol appears to specifically stimulate the in vitrocell proliferation activity of KGF-2.

Example 6 KGF-2 Gel Formulation with Citrate

[0344] The following ingredients were mixed to create a KGF-2formulation is a liquid at room temperature and that subsequently gelsupon application to skin.

[0345] 20 mM sodium citrate,

[0346] 125 mM sodium chloride,

[0347] 1 mM disodium EDTA,

[0348] 17% Pluronic 127, pH 6.0,

[0349] water.

Example 7 KGF-2 Gel Formulation with Acetate

[0350] The following ingredients were mixed to create a KGF-2formulation that can gel upon application to skin.

[0351] 20 mM sodium acetate,

[0352] 125 mM sodium chloride,

[0353] 1 mM disodium EDTA,

[0354] 17% Pluronic 127, pH 6.0,

[0355] water.

Example 8 Liquid KGF-2 Formulations

[0356] The suitability of sodium citrate as a buffer in which tomaintain KGF-2 Δ33 was evaluated in four separate formulations, and atthree separate pHs: pH 5.0, pH 5.5 and pH 6.0.

[0357] Formulations:

[0358] A. KGF-2 Δ33 1 or 2 mg/ml

[0359] 20 mM sodium citrate,

[0360] 125 mM sodium chloride,

[0361] 1 mM disodium EDTA,

[0362] water.

[0363] B. As in “A” above, further including 1% glycerol.

[0364] C. As in “A” above, further including 0.05% methionine.

[0365] D. As in “A” above, further including 1% monothioglycerol.

[0366] The concentration of KGF-2 Δ33 was 1 and 2 mg/ml in all of theabove formulations.

[0367] 2. Lyophilization Formulation

[0368] KGF-2 Δ33 was lyophilized in the presence of one of three bulkingagents: mannitol, sucrose and trehalose.

[0369] Formulations:

[0370] A. 10 mM sodium citrate, 20 mM sodium chloride, 1 mM disodiumEDTA and 4% mannitol, pH 6.0

[0371] B. 10 mM sodium citrate, 20 mM sodium chloride, 1 mM disodiumEDTA and 7% sucrose, pH 6.0

[0372] C. 10 mM sodium citrate, 20 mM sodium chloride, 1 mM disodiumEDTA and 8% trehalose, pH 6.0

[0373] The concentration of KGF-2 polypeptide was 3 mg/ml and 8 mg/ml.Evaluation parameters were RP-HPLC, SDS-PAGE, appearance, before andfollowing reconstitution with water.

[0374] 3. 10 mg/ml KGF-2 Lyophilization

[0375] Whether the formulation will permit lyophilization of the proteinat 10 mg/ml was assessed as well as the protein's subsequent stabilityafter reconstitution.

[0376] Formulation

[0377] 10 mM sodium citrate, 20 mM sodium chloride, 1 mM disodium EDTAand 4% mannitol, pH 6.0.

[0378] The lyophilized products were reconstituted with water or watercontaining 1% monothioglycerol.

[0379] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0380] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0381] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

1 33 1 627 DNA Homo sapiens CDS (1)..(624) 1 atg tgg aaa tgg ata ctg acacat tgt gcc tca gcc ttt ccc cac ctg 48 Met Trp Lys Trp Ile Leu Thr HisCys Ala Ser Ala Phe Pro His Leu 1 5 10 15 ccc ggc tgc tgc tgc tgc tgcttt ttg ttg ctg ttc ttg gtg tct tcc 96 Pro Gly Cys Cys Cys Cys Cys PheLeu Leu Leu Phe Leu Val Ser Ser 20 25 30 gtc cct gtc acc tgc caa gcc cttggt cag gac atg gtg tca cca gag 144 Val Pro Val Thr Cys Gln Ala Leu GlyGln Asp Met Val Ser Pro Glu 35 40 45 gcc acc aac tct tct tcc tcc tcc ttctcc tct cct tcc agc gcg gga 192 Ala Thr Asn Ser Ser Ser Ser Ser Phe SerSer Pro Ser Ser Ala Gly 50 55 60 agg cat gtg cgg agc tac aat cac ctt caagga gat gtc cgc tgg aga 240 Arg His Val Arg Ser Tyr Asn His Leu Gln GlyAsp Val Arg Trp Arg 65 70 75 80 aag cta ttc tct ttc acc aag tac ttt ctcaag att gag aag aac ggg 288 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu LysIle Glu Lys Asn Gly 85 90 95 aag gtc agc ggg acc aag aag gag aac tgc ccgtac agc atc ctg gag 336 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro TyrSer Ile Leu Glu 100 105 110 ata aca tca gta gaa atc gga gtt gtt gcc gtcaaa gcc att aac agc 384 Ile Thr Ser Val Glu Ile Gly Val Val Ala Val LysAla Ile Asn Ser 115 120 125 aac tat tac tta gcc atg aac aag aag ggg aaactc tat ggc tca aaa 432 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys LeuTyr Gly Ser Lys 130 135 140 gaa ttt aac aat gac tgt aag ctg aag gag aggata gag gaa aat gga 480 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg IleGlu Glu Asn Gly 145 150 155 160 tac aat acc tat gca tca ttt aac tgg cagcat aat ggg agg caa atg 528 Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln HisAsn Gly Arg Gln Met 165 170 175 tat gtg gca ttg aat gga aaa gga gct ccaagg aga gga cag aaa aca 576 Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro ArgArg Gly Gln Lys Thr 180 185 190 cga agg aaa aac acc tct gct cac ttt cttcca atg gtg gta cac tca 624 Arg Arg Lys Asn Thr Ser Ala His Phe Leu ProMet Val Val His Ser 195 200 205 tag 627 2 208 PRT Homo sapiens 2 Met TrpLys Trp Ile Leu Thr His Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15 ProGly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 ValPro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 AlaThr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly 50 55 60 ArgHis Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly 85 90 95Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu 100 105110 Ile Thr Ser Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser 115120 125 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys130 135 140 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu AsnGly 145 150 155 160 Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn GlyArg Gln Met 165 170 175 Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg ArgGly Gln Lys Thr 180 185 190 Arg Arg Lys Asn Thr Ser Ala His Phe Leu ProMet Val Val His Ser 195 200 205 3 35 DNA Homo sapiens 3 ggaccctcatgacctgccag gctctgggtc aggac 35 4 28 DNA Homo sapiens 4 ggacagccatggctggtcgt cacgttcg 28 5 29 DNA Homo sapiens 5 ggacagccat ggttcgttggcgtaaactg 29 6 31 DNA Homo sapiens 6 ggacagccat ggaaaaaaac ggtaaagttt c31 7 29 DNA Homo sapiens 7 ggacccccat ggagaactgc ccgtagagc 29 8 32 DNAHomo sapiens 8 ggacccccat ggtcaaagcc attaacagca ac 32 9 33 DNA Homosapiens 9 ggacccccat ggggaaactc tatggctcaa aag 33 10 37 DNA Homo sapiens10 ctgcccaagc ttattatgag tgtaccacca ttggaag 37 11 36 DNA Homo sapiens 11ctgcccaagc ttattacttc agcttacagt cattgt 36 12 32 DNA Homo sapiens 12gcggcacatg tcttacaacc acctgcaggg tg 32 13 28 DNA Homo sapiens 13gggcccaagc ttatgagtgt accaccat 28 14 36 DNA Homo sapiens 14 ccggcggatcccatatgtct tacaaccacc tgcagg 36 15 35 DNA Homo sapiens 15 ccggcggtaccttattatga gtgtaccacc attgg 35 16 426 DNA Homo sapiens 16 atgtcttacaaccacctgca gggtgacgtt cgttggcgta aactgttctc tttcaccaaa 60 tacttcctgaaaatcgaaaa aaacggtaaa gtttctggga ccaagaagga gaactgcccg 120 tacagcatcctggagataac atcagtagaa atcggagttg ttgccgtcaa agccattaac 180 agcaactattacttagccat gaacaagaag gggaaactct atggctcaaa agaatttaac 240 aatgactgtaagctgaagga gaggatagag gaaaatggat acaataccta tgcatcattt 300 aactggcagcataatgggag gcaaatgtat gtggcattga atggaaaagg agctccaagg 360 agaggacagaaaacacgaag gaaaaacacc tctgctcact ttcttccaat ggtggtacac 420 tcataa 426 17141 PRT Homo sapiens 17 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg TrpArg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu LysAsn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser IleLeu Glu Ile Thr Ser 35 40 45 Val Glu Ile Gly Val Val Ala Val Lys Ala IleAsn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr GlySer Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg Ile GluGlu Asn Gly Tyr Asn Thr 85 90 95 Tyr Ala Ser Phe Asn Trp Gln His Asn GlyArg Gln Met Tyr Val Ala 100 105 110 Leu Asn Gly Lys Gly Ala Pro Arg ArgGly Gln Lys Thr Arg Arg Lys 115 120 125 Asn Thr Ser Ala His Phe Leu ProMet Val Val His Ser 130 135 140 18 20 DNA Homo sapiens 18 caaccacctgcagggtgacg 20 19 78 DNA Homo sapiens 19 aacggtcgac aaatgtatgt ggcactgaacggtaaaggtg ctccacgtcg tggtcagaaa 60 acccgtcgta aaaacacc 78 20 76 DNAHomo sapiens 20 gggcccaagc ttaagagtgt accaccattg gcagaaagtg agcagaggtgtttttacgac 60 gggttttctg accacg 76 21 23 DNA Homo sapiens 21 gccacatacatttgtcgacc gtt 23 22 19 DNA Homo sapiens 22 gggcccaagc ttaagagtg 19 2323 DNA Homo sapiens 23 gccacataca tttgtcgacc gtt 23 24 90 DNA Homosapiens 24 ctgcagggtg acgttcgttg gcgtaaactg ttctccttca ccaaatacttcctgaaaatc 60 gaaaaaaacg gtaaagtttc tggtaccaag 90 25 90 DNA Homo sapiens25 agctttaaca gcaacaacac cgatttcaac ggaggtgatt tccaggatgg agtacgggca 60gttttctttc ttggtaccag aaactttacc 90 26 90 DNA Homo sapiens 26 ggtgttgttgctgttaaagc tatcaactcc aactactacc tggctatgaa caagaaaggt 60 aaactgtacggttccaaaga atttaacaac 90 27 100 DNA Homo sapiens 27 gtcgaccgttgtgctgccag ttgaaggaag cgtaggtgtt gtaaccgttt tcttcgatac 60 gttctttcagtttacagtcg ttgttaaatt ctttggaacc 100 28 25 DNA Homo sapiens 28gcggcgtcga ccgttgtgct gccag 25 29 26 DNA Homo sapiens 29 gcggcctgcagggtgacgtt cgttgg 26 30 36 DNA Homo sapiens 30 ccggcggatc ccatatgtcttacaaccacc tgcagg 36 31 34 DNA Homo sapiens 31 cgcgcgatat cttattaagagtgtaccacc attg 34 32 426 DNA Homo sapiens 32 atgtcttaca accacctgcagggtgacgtt cgttggcgta aactgttctc cttcaccaaa 60 tacttcctga aaatcgaaaaaaacggtaaa gtttctggta ccaagaaaga aaactgcccg 120 tactccatcc tggaaatcacctccgttgaa atcggtgttg ttgctgttaa agctatcaac 180 tccaactact acctggctatgaacaagaaa ggtaaactgt acggttccaa agaatttaac 240 aacgactgta aactgaaagaacgtatcgaa gaaaacggtt acaacaccta cgcttccttc 300 aactggcagc acaacggtcgacaaatgtat gtggcactga acggtaaagg tgctccacgt 360 cgtggtcaga aaacccgtcgtaaaaacacc tctgctcact ttctgccaat ggtggtacac 420 tcttaa 426 33 141 PRTHomo sapiens 33 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg LysLeu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn GlyLys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu GluIle Thr Ser 35 40 45 Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn SerAsn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser LysGlu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu AsnGly Tyr Asn Thr 85 90 95 Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg GlnMet Tyr Val Ala 100 105 110 Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly GlnLys Thr Arg Arg Lys 115 120 125 Asn Thr Ser Ala His Phe Leu Pro Met ValVal His Ser 130 135 140

What is claimed is:
 1. A pharmaceutical composition, comprising: (a) aKGF-2 polypeptide in a concentration range of about 0.02 to about 40mg/ml (w/v); (b) a buffer having a buffering capacity of between aboutpH 5.0 and about pH 8.0 at a concentration range of about 5 mM to about50 mM; and (c) a pharmaceutically acceptable diluent to bring thecomposition to a designated volume; or a reaction product thereof. 2.The pharmaceutical composition of claim 1, further comprising: (d) achelating agent at a concentration range of about 0.1 mM to about 10 mM;and (e) NaCl at a concentration range of about 0.01 mM to about 150 mM.3. The pharmaceutical composition of claim 1, further comprising one of:(f) about 0.5% to about 2% w/v glycerol, (g) about 0.1% to about 1% w/vmethionine, or (h) about 0.1% to about 2% w/v monothioglycerol.
 4. Thepharmaceutical composition of claim 1, wherein said KGF-polypeptide ispresent in a concentration range of about 0.05 to about 30 mg/ml (w/v).5. The pharmaceutical composition of claim 4, wherein saidKGF-polypeptide is present in a concentration range of about 0.1 toabout 20 mg/ml (w/v).
 6. The pharmaceutical composition of claim 5,wherein said KGF-polypeptide is present in a concentration range ofabout 0.2 to 4 mg/ml.
 7. The pharmaceutical composition of claim 1,wherein said KGF-2 polypeptide is KGF-2-Δ33.
 8. The pharmaceuticalcomposition of claim 1, wherein said diluent is water.
 9. Thepharmaceutical composition of claim 2, wherein said chelating agent isEDTA at a concentration of about 1 mM, and said NaCl is present at aconcentration of about 125 mM.
 10. The pharmaceutical composition ofclaim 1, wherein said pH is from about pH 5.5 to about pH 6.5.
 11. Thepharmaceutical composition of claim 10, wherein said pH is about pH 6.2.12. The pharmaceutical composition of claim 1, wherein said buffer isselected from the group consisting of phosphonic, acetic, aconitic,citric, glutaric, malic, succinic carbonic acid, and an alkali oralkaline earth salt thereof.
 13. The pharmaceutical composition of claim12, wherein said buffer is a phosphate, acetate or citrate salt.
 14. Thepharmaceutical composition of claim 13, wherein said buffer is a citratesalt.
 15. The pharmaceutical composition of claim 1, wherein said bufferis present in a concentration range of about 5 mM to about 30 mM. 16.The pharmaceutical composition of claim 15, wherein said buffer is acitrate salt present in a concentration of from about 10 mM to about 20mM.
 17. The pharmaceutical composition of claim 1, further comprising astabilizing amount of one or more of (a) an antioxidant or (b) athiol-compound.
 18. The pharmaceutical composition of claim 1, whereinsaid composition is maintained at a temperature at or below −20° C. 19.The pharmaceutical composition of claim 1, comprising: (a) 2 mg/ml KGF-2Δ33 polypeptide (w/v); (b) 20 mM sodium acetate; (c) 125 mM NaCl; (d) 1mM EDTA; and (e) water as diluent, or a reaction product thereof. 20.The pharmaceutical composition of claim 19, wherein said KGF-2 Δ33polypeptide is seleceted from the group consisting of KGF-2 Δ33polypeptide having an N-terminal methionine, KGF-2 Δ33 polypeptidelacking an N-terminal methionine, and a mixture thereof.
 21. Apharmaceutical composition, comprising: (a) a KGF-2 polypeptide in aconcentration range of about 0.02 to about 40 mg/ml (w/v); (b) a bufferhaving a buffering capacity of between about pH 5.0 and about pH 8.0 ata concentration range of about 5 mM to about 50 mM; (c) a bulking agent;and (d) a pharmaceutically acceptable diluent to bring the compositionto a designated volume; or a reaction product thereof.
 22. Thepharmaceutical composition of claim 21, wherein said bulking agent isselected from the group consisting of sucrose, glycine, mannitol,trehalose, and mixtures thereof.
 23. The pharmaceutical composition ofclaim 21, further comprising: (e) a chelating agent at a concentrationrange of about 0.1 mM to about 10 mM; and (f) NaCl at a concentrationrange of about 0.01 mM to about 125 mM.
 24. The pharmaceuticalcomposition of claim 22, wherein said bulking agent is sucrose or amixture of sucrose and glycine.
 25. The pharmaceutical composition ofclaim 22, wherein said bulking agent is present in a concentration ofabout 2% to about 10% w/v.
 26. The pharmaceutical composition of claim22, wherein said bulking agent is 5% mannitol, 7% sucrose, 8% trehalose,or 2% glycine+0.5% sucrose.
 27. The pharmaceutical composition of claim21, wherein said pH is about pH 6.2.
 28. The pharmaceutical compositionof claim 21, wherein said diluent is water.
 29. The pharmaceuticalcomposition of claim 21, wherein said buffer is selected from the groupconsisting of phosphonic, acetic, aconitic, citric, glutaric, malic,succinic carbonic acid, and an alkali or alkaline earth salt thereof.30. The pharmaceutical composition of claim 29, wherein said buffer is aphosphate or citrate salt.
 31. The pharmaceutical composition of claim30, wherein said buffer is a citrate salt.
 32. The pharmaceuticalcomposition of claim 28, wherein over 90% of the water is removed bylyophilization.
 33. The pharmaceutical composition of claim 32, which isreconstituted in with an amount of sterile water effective to maintainisotonic conditions of about 290 mOsm.
 34. The pharmaceuticalcomposition of claim 21, wherein said KGF polypeptide is KGF-2-Δ33. 35.The pharmaceutical composition of claim 34, wherein said KGF-2 Δ33polypeptide is seleceted from the group consisting of KGF-2 Δ33polypeptide having an N-terminal methionine, KGF-2 Δ33 polypeptidelacking an N-terminal methionine, and a mixture thereof.
 36. Thepharmaceutical composition of claim 21, wherein said buffer is added ina concentration from about 5 mM to about 50 mM
 37. The pharmaceuticalcomposition of claim 36, wherein said buffer is citrate at aconcentration of about 10 mM.
 38. The pharmaceutical composition ofclaim 21, further including a stabilizing amount of one or more of (g)an antioxidant, or (h) a thiol-compound.
 39. The pharmaceuticalcomposition of claim 32, wherein said composition is reconstituted insterile water containing a stabilizing amount of an antioxidantcomprising: a) about 0.01% to about 2% w/v monothioglycerol, b) about0.01% to about 2% w/v ascorbic acid, c) about 0.01% to about 2% w/vmethionine or d) combinations thereof.
 40. A pharmaceutical composition,comprising: (a) a KGF-2 polypeptide in a concentration range of about0.02 to about 40 mg/ml (w/v); (b) citric acid or a pharmaceuticallyacceptable salt thereof, at a concentration range of about 5 mM to about20 mM; (c) NaCl at a concentration range of about 0.01 mM to about 125mM, (d) EDTA at a concentration range of about 0.1 mM to about 10 mM and(e) one or more of sucrose, mannitol, glycine or trehalose at aconcentration range of about 2% w/v to about 15% w/v; and (f) water. 41.The pharmaceutical composition of claim 40, wherein said KGF-2polypeptide is present at a concentration of about 2 mg/ml, about 4mg/ml, or about 10 mg/ml.
 42. The pharmaceutical composition of claim40, wherein over 90% of the water is removed by lyophilization.
 43. Thepharmaceutical composition of claim 1, further comprising a thickeningagent in an amount effective to raise the viscosity to about 50 to about10,000 cps.
 44. The pharmaceutical composition of claim 43, wherein saidthickening agent is present in an amount effective to raise theviscosity to about 50 to about 1,000 cps.
 45. The pharmaceuticalcomposition of claim 44, wherein said thickening agent in an amounteffective to raise the viscosity to about 200 to about 300 cps.
 46. Thepharmaceutical composition of claim 21, further comprising a thickeningagent in an amount effective to raise the viscosity to about 50 to about10,000 cps.
 47. The pharmaceutical composition of claim 43, wherein said5 thickening agent is present in a concentration of 0 to 5% (w/w). 48.The pharmaceutical composition of claim 43, wherein said thickeningagent is a water soluble etherified cellulose or a high molecular weightpolymer of acrylic acid cross-linked with allylsucrose or an allyl etherof pentaerythritol.
 49. The pharmaceutical composition of claim 48,wherein said etherified cellulose is an alkyl cellulose, hydroxyalkylcellulose, carboxyalkyl cellulose or alkylhydroxyalkyl cellulose. 50.The pharmaceutical composition of claim 43, wherein said etherifiedcellulose is methylcellulose, hydroxyethyl cellulose, hydroxy propylcellulose, hydroxy propyl methylcellulose, or carboxymethyl cellulose.51. The pharmaceutical composition of claim 49, wherein said etherifiedcellulose derivative has a molecular weight of about 50,000 to about700,000 and is present in a concentration of about 0 to about 20% byweight.
 52. The pharmaceutical composition of claim 51, wherein saidetherified cellulose derivative has a molecular weight of about 80,000to about 240,000 and is present in a concentration of about 2% to about8% by weight.
 53. The pharmaceutical composition of claim 46, whereinsaid buffer is citrate in a concentration of about 10 mM to about 50 mM.54. The pharmaceutical composition of claim 53, wherein said buffer iscitrate in a concentration of about 10 mM to about 20 mM citrate. 55.The pharmaceutical composition of claim 53, wherein said bulking agentis sucrose in a concentration of about 0.01% to about 5% sucrose. 56.The pharmaceutical composition of claim 55, wherein said thickeningagent is added directly to a liquid formulation and thereafterlyophilized.
 57. The pharmaceutical composition of claim 55, whereinsaid thickening agent is added to a lyophilized formulation byreconstituting said formulation by adding a suitable diluent having athickening agent dissolved therein.
 58. A thickened KGF-2 polypeptidesolution composition formed by mixing: (a) a topically effective amountof a KGF-2 polypeptide; (b) about 10 mM to about 500 mM sodium citratebuffer; (c) about 0.01 to about 150 mM NaCl; (d) 1 mM EDTA; (e) about0.1 to about 7% sucrose; (f) about 0.75 to about 1.5% (w/w) carboxymethyl cellulose or about 0.5 to about 1.5% hydroxy propyl methylcellulose or about 0.25 to about 0.75% hydroxy ethyl cellulose or about0 to 1% carbomer or any combination thereof.
 59. The composition ofclaim 1, further comprising a gelling agent in an amount effective toraise the viscosity to about 0.1 to about 10,000 cps at roomtemperature.
 60. The composition of claim 21, further comprising agelling agent in an amount effective to raise the viscosity to about 0.1to about 10,000 cps at room temperature.
 61. The composition of claim59, wherein said gel forming agent is a water-soluble polymer capable offorming a viscous aqueous solution, or non-water soluble,water-swellable polymer capable of forming a viscous solution.
 62. Thecomposition of claim 61, wherein said gel forming agent is a highmolecular weight polymer selected from the group consisting of vinylpolymer, polyoxyethylene-polyoxypropylene copolymer, polysaccharide,protein, poly(ethylene oxide), acrylamide polymer or a salt thereof. 63.The composition of claim 62, wherein said gel forming agent is (1) avinyl polymer selected from the group consisting of polyacrylic acid,polymethacrylic acid, polyvinyl pyrrolidone polyvinyl alcohol, and saltsand esters thereof; or (2) a polysaccharide selected from the groupconsisting of a cellulose derivative, a glycosaminoglycan, agar, pectin,alginic acid, dextran, α-amylose, amylopectin, chitosan, and saltsesters thereof.
 64. The composition of claim 62, wherein said gelforming agent is a glycosaminoglycan selected from the group consistingof hyaluronic acid, chondroitin, chondroitin-4-sulfate, heparan sulfate,heparin and salts and esters thereof.
 65. The composition of claim 64,wherein said glycosaminoglycan is present in combination with collagen,gelatin, or fibronectin.
 66. The composition of claim 62, wherein saidgel forming agent is an acrylamide polymer selected from the groupconsisting of a polyacrylamide or a polymethacrylamide.
 67. Thecomposition of claim 62, wherein said gel forming agent is apolyoxyethylene-polyoxypropylene block copolymer.
 68. The composition ofclaim 67, which comprises about 10 to about 60% by weight of apolyoxyethylene-polyoxypropylene block copolymer having an averagemolecular weight of about 500 to 50,000.
 69. The composition of claim68, which comprises about 14 to about 18% by weight of apolyoxyethylene-polyoxypropylene block copolymer having a molecularweight in the range 1,000 to 15,000.
 70. The composition of claim 69,wherein said gel forming agent is a polyoxyethylene-polyoxypropyleneblock copolymer is Pluronic F108, Pluronic F127, or Poloxamer
 407. 71.The composition of claim 1, wherein said KGF-2 polypeptide is present ina concentration of about 0.01 mg/ml to about 10 mg/ml.
 72. Thecomposition of claim 59, wherein said composition is formed by mixing:(a) a KGF-2 polypeptide, in a final calculated concentration of 0.01mg/ml to about 10 mg/ml; (b) an effective amount of a buffering agent;(c) about 10% to about 60% by weight of apolyoxyethylene-polyoxypropylene block copolymer having an averagemolecular weight of about 500 to 50,000; and (d) a pharmaceuticallyacceptable diluent, preferably water.
 73. The composition of claim 72,wherein polyoxyethylene-polyoxypropylene block copolymer is present at aconcentration of about 14% to about 18%.
 74. A KGF-2 gel formulation,comprising: (a) a pharmaceutically active amount of KGF-2 polypeptide;(b) about 10 mM to about 500 mM sodium citrate; (c) about 0.01 mM toabout 150 mM NaCl; (d) about 1 mM EDTA; (e) about 0.1% to about 7%sucrose; (f) about 14% to about 18% Pluronic F127; and (g) about pH 6.275. A KGF-2 gel formulation, comprising: (a) a KGF-2 polypeptide at aconcentration range of about 0.01 mg/ml to about 10 mg/ml (w/v), (b)sodium citrate at a concentration range of about 5 mM to about 20 mM;(c) about 10% to about 25% (w/v), of Pluronic 127 or Poloxamer 407; and(d) water to volume.
 76. The gel formulation of claim 75, furthercomprising: (a) EDTA at a concentration range of about 0.1 mM to about10 mM. (b) NaCl at a concentration range of about 0.01 mM to about 125mM.
 77. The pharmaceutical composition of claim 1, wherein said KGF-2polypeptide is a N-terminal deletion selected from the group consistingof Ala (63)-Ser (208) (KGF-2Δ28) and Ser (69)-Ser (208) (KGF-2Δ33). 78.The pharmaceutical composition of claim 77, wherein said KGF-2polypeptide has an N-terminal methionine, lacks an N-terminalmethionine, or is a mixture thereof.
 79. The pharmaceutical compositionof claim 1, wherein said KGF-2 polypeptide is a N-terminal or C-terminaldeletion mutant selected from the group consisting of Ala (39)-Ser(208); Pro (47)-Ser (208); Val (77)-Ser (208); Glu (93)-Ser (208); Glu(104)-Ser (208); Val (123)-Ser (208); Gly (138)-Ser (208); Met (1), Thr(36); and Cys (37)-Lys (153).
 80. The pharmaceutical composition ofclaim 79, wherein said KGF-2 polypeptide has an N-terminal methionine,lacks an N-terminal methionine, or is a mixture thereof.
 81. Thepharmaceutical composition of claim 7, wherein said KGF-2 Δ33polypeptide is selected from the group consisting of KGF-2 Δ33polypeptide having an N-terminal methionine, KGF-2 Δ33 polypeptidelacking an N-terminal methionine, and a mixture thereof.